Palladium catalyzed nucleoside modification methods using nucleophiles and carbon monoxide

ABSTRACT

This invention discloses a method for the preparation modified nucleosides and nucleotides using a palladium catalyst, a nucleophile and carbon monoxide.

FIELD OF THE INVENTION

This invention relates to the field of nucleic acid chemistry,specifically to a process for preparing modified nucleosides. Thenucleosides can be pyrimidines or purines. The pyrimidine compounds ofthe invention can be modified at the 5-, or 6-position of the pyrimidinering. The purine compounds of the invention can be modified at the 2-,6- or 8-position of the purine ring. Most preferably, the inventionincludes a process for preparing 8-position modified purine compoundsand 5-position modified pyrimidine compounds. The present invention alsoincludes the modified nucleosides produced by the method. The inventionalso includes the use of the modified nucleosides as anti-viral,anti-bacterial, anti-fungal or anti-neoplastic agents or as part of anoligonucleotide.

BACKGROUND OF THE INVENTION

Until quite recently, the consideration of oligonucleotides in anyfunction other than strictly informational was not known. Despite thefact that certain oligonucleotides were known to have interestingstructural possibilities (e.g., t-RNAs) and other oligonucleotides werebound specifically by polypeptides in nature, very little attention hadbeen focused on the non-informational capacities of oligonucleotides.For this reason, among others, little consideration had been given tousing oligonucleotides as pharmaceutical compounds.

There are currently at least three areas of exploration that have led toserious studies regarding the use of oligonucleotides aspharmaceuticals. In the most advanced of the fields, antisenseoligonucleotides are utilized to bind to certain regions in an organismto prevent the expression of proteins or to block various cellfunctions. The discovery of RNA species with catalyticfunctions--ribozymes--has led to the consideration of RNA species thatserve to perform intracellular reactions that will achieve desiredeffects. And lastly, the discovery of the SELEX process (SystematicEvolution of Ligands by EXponential Enrichment) has shown the researchcommunity that oligonucleotides can be identified that will bind toalmost any biologically interesting target.

The use of antisense oligonucleotides as a method for controlling geneexpression and the potential for using oligonucleotides aspharmaceutical materials has prompted investigations into theintroduction of a number of chemical modifications into oligonucleotidesto increase their therapeutic activity. Such modifications are designedto increase cell penetration of the oligonucleotides, to stabilize themfrom nucleases and other enzymes that degrade or interfere with thestructure or activity of the oligonucleotide analogs in the body, toenhance their binding to targeted nucleic acids, to provide a mode ofdisruption (terminating event) once sequence-specifically bound totargeted nucleic acids, and to improve their pharmacokinetic properties.For example, PCT Patent Application Publication WO 91/14696, entitled:Oligonucleotide-Transport Agent Disulfide Conjugates, describes a methodfor chemically modifying antisense oligonucleotides to enhance entryinto a cell.

A variety of methods have been used to render oligonucleotides resistantto degradation by exonucleases. PCT Patent Application Publication WO90/15065, entitled: Exonuclease-Resistant Oligonucleotides and Methodsfor Preparing the Same, describes a method for makingexonuclease-resistant oligonucleotides by incorporating two or morephosphoramidite and phosphoromonothionate and/or phosphorodithionatelinkages at the 5' and/or 3' ends of the oligonucleotide. PCT PatentApplication Publication WO 91/06629, entitled: Oligonucleotide Analogswith Novel Linkages, describes oligonucleotide compounds with one ormore phosphodiester linkages between adjacent nucleotides replaced by aformacetal/ketal type linkage which are capable of binding RNA or DNA.

A common strategy for stabilization of RNA against endonucleolyticcleavage is to modify the 2'-position of ribonucleotides. Interferencewith base recognition by enzymes can be used to approach stabilizationagainst base-specific endonucleolytic cleavage. Several strategies forthis modification are known, including modification with 2'-amino and2'-fluoro (Hobbs et al. (1973) Biochemistry 12:5138; Guschlbauer et al.(1977) Nucleic Acids Res. 4:1933), and 2'-OCH₃ (Shibahara et al. (1987)15:4403; Sproat et al. (1989) Nucleic Acids Res. 17:3373). PCT PatentApplication Publication WO 91/06556, entitled: 2' ModifiedOligonucleotides, describes nuclease-resistant oligomers withsubstituents at the 2' position. PCT Patent Application Publication WO91/10671, entitled: Compositions and Methods for Detecting andModulating RNA Activity and Gene Expression, describes antisenseoligonucleotides chemically modified at the 2' position and containing areactive portion capable of catalyzing, alkylating, or otherwiseeffecting the cleavage of RNA, a targeting portion, and a tether portionfor connecting the targeting and reactive portions.

The 5-position of pyrimidines may also be chemically modified. Theintroduction of modifications at the C-5 position of pyrimidines may beenvisioned to interfere with the recognition by pyrimidine specificendonucleases. However, this concept is not as clear cut as themodification of the 2'-position of ribonucleotides.

The use of palladium to catalyze carbon-carbon bond formation at the 5position of pyrimidine nucleosides is known. A superior method for5-position modification of pyrimidines is described in U.S. Pat. No.5,428,149, entitled "Method for Palladium Catalyzed Carbon-CarbonCoupling and Products," now U.S. Pat. No. 5,428,149, which is hereinincorporated by reference in its entirety. The first examples of5-position pyrimidine modifications were demonstrated by Bergstrom(Bergstrom et al. (1976) J. Am. Chem. Soc. 98:1587, (1978) J. Org. Chem.43:2870, (1981) J. Org. Chem. 46:1432 and 2870, (1982) J. Org. Chem.47:2174) and Daves (Arai and Daves (1978) J. Am. Chem. Soc., 100:287;Hacksell and Daves (1983) J. Org. Chem. 48:2870). Bergstrom and Davesused 5-mercurial-deoxyuridine compounds, the same as those used byDreyer and Dervan ((1985) Proc. Natl. Acad. Sci. USA 82:968), to tetherfunctional groups to oligonucleotides.

One method for simple carbon-carbon coupling reactions to the 5-positionof uridines is described in the work of Crisp (1989) Syn. Commun.19:2117. Crisp forms deoxyuridines functionalized at the 5-position byreacting protected 5-iodo-2'-deoxyuridine with alkenylstannanes inacetonitrile in the presence of a Pd (II) catalyst.

To date, very little work has been done to modify purine nucleosidesusing palladium catalysis. Van Aerschot et al., ((1993) J. Med. Chem36:2938-2942) report that 2-, 6-, and 8-halogenated adenosines can bemodified with symmetric organotin reagents. However, symmetric organotincompounds are not widely available. Sessler et al., ((1993) J. Am. Chem.115:10418-10419) describe the arylation of protected 8-bromoguanosinewith 4-tributyltinbenzaldehyde. However, using this procedure, asignificant amount of starting material (28%) was unreacted. A superiormethod for modifying purine nucleosides using palladium catalysts isdescribed in U.S. patent application Ser. No. 08/347,600, filed Dec. 1,1994, entitled "Purine Nucleoside Modification by Palladium CatalyzedMethods", which is herein incorporated by reference in its entirety.

Additionally, very little work has been done in the area of palladiumcatalyzed amidations. Schoenberg, et al. (J. Org. Chem. (1974) 39:3327)describe amidation of aryl and alkenyl halides, however, this work doesnot include nucleoside substrates or the use of a PdL₄ catalyst.

SELEX (Systematic Evolution of Ligands for EXponential Enrichment) is amethod for identifying and producing nucleic acid ligands, termed"nucleic acid antibodies", e.g., nucleic acids that selectively bind totarget molecules (Tuerk and Gold (1990) Science 249:505). The methodinvolves selection from a mixture of candidates and step-wise iterationsof structural improvement, using the same general selection theme, toachieve virtually any desired criterion of affinity and selectivity.Starting from a mixture of nucleic acids, the method includes steps ofcontacting the mixture with the target under conditions favorable forinteraction, partitioning non-interacting nucleic acids from thosenucleic acids which have interacted with the target molecules,dissociating the nucleic acid-target pairs, amplifying the nucleic acidsdissociated from the nucleic acid-target pairs to yield a mixture ofnucleic acids enriched for those which interact with the target, thenreiterating the steps of interacting, partitioning, dissociating andamplifying through as many cycles as desired.

The methods of the present invention may be combined with SELEX toproduce nucleic acids containing modified nucleotides. The presence ofmodified nucleotides may result in nucleic acids with an alteredstructure exhibiting an increased capacity to interact with targetmolecules. The steric and electronic influence of modified nucleosidesmay also act to prevent nuclease degradation. Incorporation of modifiednucleotides into oligonucleotides is well known to those skilled in theart (Dewey, T. et al., J. Amer. Chem. Soc. (1995) 117:8474-8475; WalkerG. C. et al., Biochemistry (1975) 14:817-823; Connolly, B. A., pp155-183 In Oligonucleotides and Analogues: A Practical Approach (1991)(editor F. Eckstein) IRL Press, New York).

BRIEF SUMMARY OF THE INVENTION

The present invention includes a novel method for introducing chemicalmoieties at various positions of nucleoside rings utilizing a palladiumcatalyst and a nucleophile and carbon monoxide. Preferably, themodifications are at the 5- or 6-position of a pyrimidine ring or at the2-, 6-, or 8-positions of the purine ring. Most preferably themodifications are at the 5-position of the pyrimidine ring and at the8-position of the purine ring. Particularly preferred modifications ofthe nucleoside ring include the introduction of an amide or estermoiety. For the preferred modifications, the nucleophile is a primary orsecondary amine.

This invention includes a reaction scheme for producing a wide varietyof modified nucleoside molecules. A key element in the production of themodified nucleosides is the use of a palladium catalyst in conjunctionwith a nucleophile and carbon monoxide.

More specifically, the invention provides a method for the preparationof a modified nucleoside comprising the steps of reacting a nucleosidestarting material containing a leaving group attached to a carbon atomof the nucleoside starting material with a nucleophile and carbonmonoxide in the presence of a palladium catalyst; and isolating themodified nucleoside. The modified nucleosides produced by this methodare also included in the invention.

This invention further includes a method of preparing stabilized nucleicacids wherein the modified nucleoside is coupled to a sugar modified atthe 2'-position or the 3'-position.

The modified nucleosides of the invention have many uses including, butnot limited to, use as anti-viral, anti-bacterial, anti-fungal, oranti-neoplastic agents and use as part of an oligonucleotide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a method for modifying a nucleoside ringby reacting a nucleoside starting material with a nucleophile and carbonmonoxide in the presence of a palladium catalyst. The invention includesthe modifications of both pyrimidines and purines. The pyrimidines havethe following structures and conventional numbering: ##STR1## Thepyrimidine ring can be modified at the 5- or 6-position; most preferablythe 5-position is modified. The purines have the following structuresand conventional numbering: ##STR2## The purine can be modified atpositions 2-, 6- and 8- of the purine ring; most preferably the8-position is modified. Introduction of a variety of modifications tothe nucleoside ring are contemplated by this invention. However,particularly preferred modifications to the nucleoside ring include theintroduction of an amide or ester moiety. In the preferredmodifications, the nucleophile for the carboxyamidation reaction is aprimary or secondary amine.

The present invention extends to all novel compounds that can beprepared according to the methods of the present invention. The presentinvention also includes oligonucleotides that contain one or more of thenovel substituted nucleosides of this invention. The present inventionalso includes the use of the modified nucleosides in variouspharmaceutical areas, particularly as anti-virals, anti-bacterials,anti-fungals and anti-neoplastics.

The general reactions of the present invention can be characterized asfollows: ##STR3##

As used herein the term "modified nucleoside" is intended to encompassany nucleoside base, nucleoside, or nucleotide that may be prepared bythe method of the present invention. The terms nucleoside base,nucleoside and nucleotide can be used interchangeably herein. Themodified nucleosides of the invention can contain various othermodifications on the base and sugar.

"Nucleoside starting material" is defined herein as any nucleoside base,nucleoside or nucleotide which has an attached acceptable leaving group(X). Nucleoside starting materials include all nucleosides, bothnaturally occurring and non-naturally occurring. Preferably, nucleosidestarting materials include purines and pyrimidines, which includeuracil, thymine, cytosine, adenine and guanine starting materials, orprotected derivatives thereof. In certain embodiments, the protectedderivatives include those wherein R" is dimethyl formamidine or an acylgroup, preferably selected from the group consisting of isobutyryl,acetyl, phenoxyacetyl, and benzoyl. R" is H in unprotected nucleosides.The leaving group can be attached to any free carbon on the nucleosidestarting material. The acceptable leaving group is displaced during thecatalysis reaction and replaced by C(O)YR chemical moieties to yield themodified base, nucleoside or nucleotide of the invention. The nucleosidestarting material can have a sugar moiety attached in the form of aribose, deoxyribose, dideoxyribose and any combination of 2', 3' or 5'modifications thereof. The invention contemplates the above sugarmoieties and any suitable derivatives thereof, such as a ribose or2'-deoxyribose wherein the hydroxyl groups have been partially or fullyprotected. For example, the 5'-hydroxyl can be present as the mono-,di-, or tri-phosphate.

"Pyrimidine starting material" is defined herein as a pyrimidine base,pyrimidine nucleoside or pyrimidine nucleotide which has an attachedacceptable leaving group (X). Pyrimidine starting materials include allpyrimidines, both naturally occurring and non-naturally occurring.Preferably, pyrimidine starting materials include uracil, thymine, andcytosine, or protected derivatives thereof. The leaving group can beattached to any free carbon on the base of the nucleoside, preferably atthe 5- or 6-position. The most preferred attachment is at the 5-positionof the pyrimidine ring. The acceptable leaving group is displaced duringthe catalysis reaction and replaced by C(O)YR chemical moieties to yieldthe modified pyrimidine. The pyrimidine starting material can have asugar moiety attached in the form of a ribose, deoxyribose,dideoxyribose and any combination of 2', 3' or 5' modifications thereof.The invention contemplates the above sugar moieties and any suitablederivatives thereof, such as a ribose or 2'-deoxyribose wherein thehydroxyl groups have been partially or fully protected. For example, the5'-hydroxyl can be present as the mono-, di-, or tri-phosphate.

"Purine starting material" is defined herein as a purine base, purinenucleoside or purine nucleotide which has an attached acceptable leavinggroup (X). Purine starting materials include adenine and guaninestarting materials, or protected derivatives thereof. The leaving groupcan be attached to any carbon atom of the base of the purine, preferablyat the 2-, 6-, or 8-position of the purine ring. The most preferredattachment is at the 8-position. The acceptable leaving group isdisplaced during the catalysis reaction and replaced by C(O)YR chemicalmoieties to yield the modified purine. The purine starting material canhave a sugar moiety attached in the form of a ribose, deoxyribose,dideoxyribose and any combination of 2', 3' or 5' modifications thereof.The invention contemplates the above sugar moieties and any suitablederivatives thereof, such as a ribose or 2'-deoxyribose wherein thehydroxyl groups have been partially or fully protected. For example, the5'-hydroxyl can be present as the mono-, di-, or tri-phosphate.

"Acceptable leaving group" is defined herein as a group which is asuitable counterion for palladium(II), and is designated herein as X. Inthe most general embodiments of this invention, X is any of a number ofacceptable leaving groups well known to those skilled in the art.Acceptable leaving groups include, but are not limited to, acetate,trifluoroacetate, trifluoromethyl sulfonate, tosylate, methane sulfonateand boronic esters and acids. In the preferred embodiment, X is ahalogen, and in the most preferred embodiment X is bromine or iodine.The leaving group is attached to the carbon atom of the purine startingmaterial by methods known to one of ordinary skill in the art.

"Nucleophile" is defined herein as would be understood by one ofordinary skill in the art. Specifically, a nucleophile is an electronrich chemical moiety capable of displacing a leaving group. Due to thenature of the catalytic reaction, the CO is inserted between saidnucleoside starting material and said nucleophile. Anyone skilled in theart would recognize a useful nucleophile which could be used in anucleophilic substitution reaction. Examples of preferred nucleophilesinclude, but are not limited to, amines, alcohols, and thiols.

In a preferred embodiment, the general structure of the nucleophilesused in the present invention is RYH, where Y=O, S, NH, or NR'. R and R'can optionally be part of a ring-structure, which can be aromatic,aliphatic or heterocyclic. In the preferred embodiments of the inventionthe nucleophile (RYH) is selected from the group consisting of aliphaticor aromatic, primary or secondary amines (including cyclic amines),alcohols and thiols; wherein R and R' are selected from the groupconsisting of substituted or unsubstituted C1-C20 alkyl (straight-chainor branched), C2-C20 alkenyl (straight-chain or branched), aryl,heterocyclic, and natural and unnatural amino acids.

In a preferred embodiment, the nucleophile has the structure RYH,wherein,

Y is selected from the group consisting of O, S, and NH;

R is (CH_(z))_(m) (CH₃)_(n) wherein z is 0, 1, or 2; m is 0-19; n is 0,1, 2, or 3; and wherein one or more of the H are optionally substitutedwith ═O, --OH, ═NH, NH₂, +NMe₃ Cl, ##STR4## or an amino acid.

In the most preferred embodiments of the invention, the nucleophiles areselected from the following group: ##STR5##

The R and R' groups of the nucleophile can include various functionalgroups which can be used to introduce a broad array of functionalcapabilities to the nucleosides prepared by this method. The nucleophilefunctional groups can include, among others: amides, esters, nitriles,nitros, ureas, halides, cyanates, alcohols, amines, ethers, thiols, arylsubstituents, etc. as recognized by those of ordinary skill in the art.Any replacement of a hydrogen or functional group on the nucleophile isreferred to as a "substitution" for the purposes of definition.

The palladium catalyst of the present invention may be characterizedmost generally as PdL₄ or PdL₃, where L is one of any number of commonlyemployed ligands of palladium. The palladium catalyst can be pre-made(e.g., PdL₄, wherein L is triphenyl phosphine, etc.) or made in situfrom Pd(0) or Pd(II) and phosphine ligands as is known to one ofordinary skill in the art (e.g., bis(benzylideneacetone)Pd(0)!,Pd(OAc)₂, etc.). PdL₄ is the preferred palladium catalyst of theinvention. It is within the skill and knowledge of those skilled in theart to recognize the various ligands that may be employed. Examples ofcommon ligands (L) include, but are not limited to, PPh₃, (o-tol)₃ P,P(p-C₆ H₄ SO₃ Na)₃, CH₃ CN, DMSO, N,N-dimethylformamide ##STR6## In thepreferred embodiments of the catalytic species of this invention L isPPh₃ (triphenyl phosphine, or P(C₆ H₅)₃) or P(p-C₆ H₄ SO₃ Na)₃. Thepreparation of certain catalysts of the present invention is describedin U.S. Pat. No. 5,428,149, filed Jun. 14, 1993, entitled "Method forPalladium Catalyzed Carbon-Carbon Coupling and Products" which isincorporated by reference herein.

In certain embodiments, it may be advantageous to include additionalbasic, non-nucleophilic components in the reaction. Examples ofdesirable bases include, but are not limited to, Et₃ N, EtN(iPr)₂,1,8-diazabicyclo 5.4.0!undec-7-ene (DBU), and 1,4-diazabicyclo2.2.2!octane (DABCO). Acceptable solvents for the reaction includeacetonitrile, dioxane, acetone, ethyl acetate, benzene, dimethylformamide, dimethyl acetamide, dimethyl sulfoxide, water, THF,hexamethylphosphoramide (HMPA), and hexamethylphosphoroustriamide(HMPT). The temperature ranges for the reaction typically are between 60and 100 degrees centigrade, however, other suitable temperature rangesare also contemplated.

The following reactant concentrations and reaction conditions are usefulin the preferred embodiments of the present invention. The nucleophileis used preferably in the range from 0.0 to 2.0 M. The concentration ofthe palladium catalyst can range from 0.0005 to 0.2 M. The CO pressurecan range from 10 to 1000 psi. The concentration of the nucleosidestarting material can range from 0.010 to 1.0 M.

The modified nucleosides and nucleotides of the present invention arecontemplated for use in oligonucleotides. Standard techniques forincorporation of nucleosides into oligonucleotides can be used with themodified nucleosides of the invention. Typically, the oligonucleotidesof the invention are less than 500 bases, usually less than 100 bases,and most preferably less than 50 bases. The modified nucleosides aresuitable for in vitro transcription procedures. The oligonucleotidescontaining the modified nucleosides have a number of various utilities.Specifically, the oligonucleotides interact with biological targets orhave facilitating properties. The oligonucleotides can be useful invarious diagnostic applications as well.

The nucleosides or nucleotides may also show antineoplastic,antibacterial, antifungal or antiviral activity. The nucleosides andnucleotides may also demonstrate other therapeutic properties. Standardassays are known to one of ordinary skill for determination of suchactivities. Formulation and administration routes are well known tothose of ordinary skill in the art. Additionally, prodrug technology canbe used as a delivery system for the nucleosides and nucleotides of theinvention. Particularly, the nucleosides or nucleotides can be attachedto lipids to improve pharmacology and oral availability, among othercharacteristics. Specifically, 5'-diacylglycero- ordialkylglycerophosphate-derivatives of the nucleosides and nucleotidesof the invention are useful. These modified nucleosides and nucleotidesare particularly interesting for antiviral applications. Thediacylglycerophosphates of nucleosides and non-nucleosides have beenused for modulation of pharmacokinetic behavior, modulation ofbioavailability, and modulation of toxicity as described in U.S. Pat.No. 5,223,263 which is herein incorporated by reference.

Stability towards endo-nucleolytic degradation in serum can be achievedby introducing 2'-deoxy-2'-fluoro- or 2'-deoxy-2'-aminonucleosides tothe pyrimidine positions of the ligand (Pieken et al. (1991) Science253:314). The modified nucleosides of the present invention may also becoupled with 2' substituted species that would also be useful in avariety of situations. The incorporation of halogenated nucleosides mayalso prove valuable for enhanced ligand-target interaction.

EXAMPLES

The following examples are illustrative of preferred embodiments ofmethods of preparation and products of the invention and are not to beconstrued as limiting the invention thereto.

EXAMPLE 1 Purine Modifications with Amines

The following general procedures were employed to produce the modifiedpurines of Table I.

The general scheme: ##STR7## The more specific scheme: ##STR8## Thevarious nucleoside base starting materials that can be used in the morespecific scheme: ##STR9##

                                      TABLE I                                     __________________________________________________________________________    Examples of modified purine nucleosides.                                               Amine Nucleophile          Product                                                                           Isolated                              Entry                                                                            Nucleoside                                                                          H.sub.2 NR                 ID  Yield (%)                             __________________________________________________________________________    1  1                                                                                   2                           4  .sup. 87.sup.b                        2  1                                                                                   3 #STR10##                  5  95                                    3  2                                                                                   3 #STR11##                  6  93                                    4  2                                                                                   4 #STR12##                  7  .sup.  84.sup.a                       5  2                                                                                   5 #STR13##                  8  95                                    6  2                                                                                   6 #STR14##                  9  98                                    7  2                                                                                   7 #STR15##                 10  41                                    8  2                                                                                   8 #STR16##                 11  91                                    9  2                                                                                   9 #STR17##                 12  .sup.  62.sup.c                       10 2                                                                                   0 #STR18##                 13  71                                    11 3                                                                                   6 #STR19##                 14  85                                    12 3                                                                                   8 #STR20##                 15  98                                    13 3                                                                                   0 #STR21##                 16  88                                    14 3                                                                                   3 #STR22##                 17  81                                    15 3                                                                                   1 #STR23##                 18  56                                    __________________________________________________________________________     .sup.a The reaction produced 16% of a side product with no CO insertion       and direct coupling of the nucleophile to the nucleoside starting             material.                                                                     .sup.b The reaction produced 15% of a side product with no CO insertion       and direct coupling of the nucleophile to the nucleoside starting             material.                                                                     .sup.c The reaction produced 14% of a side product with no CO insertion       and direct coupling of the nucleophile to the nucleoside starting materia     and 14% of a product resulting from CO insertion and the hydroxide servin     as nucleophile rather than the amine.                                    

The following general procedures were followed to produce the modifiedpurine nucleosides of Table I.

General. The ¹ H and ¹³ C NMR spectra were obtained in CD₃ OD, D₂ O,CDCl₃, or DMSO-d⁶ on a Bruker ARX-300 spectrometer using the deuteratedsolvent as an internal standard. Positive ion fast atom bomdardment massspectra (FAB⁺) were performed at the Univ. of California at BerkeleyMass Spec. facility.

Materials. 8-Bromoadenosine, 8-bromoguanosine dihydrate, morpholine,n-butylamine, isopropylamine, tert-butylamine, aniline,4-aminomethylpyridine, (2-aminoethyl)trimethyl-ammonium chloride,arginine ethyl ester, ethanolamine, triethylamine, DMF andN,N-dimethylacetamide (DMA) were purchased from Aldrich Chemical Companyand were used as received unless otherwise noted.N-(2-Aminoethyl)biotinamide hydrobromide was purchased from MolecularProbes, Inc.

General procedure for palladium catalyzed coupling reaction. To a glassbomb with a Teflon valve was added the nucleoside specified in Table 1(0.5 mmol), the amine nucleophile specified in Table 1 (1.0 mmol),Pd(PPh₃)₄ (0.0015 mmol), triethylamine (1.0 mmol) and DMF (or DMA). Theglass bomb was evacuated and charged with CO (50 psi), then heated tothe desired temperature for 24 hours. The solvent was removed and theresidue was purified by flash chromatography on silica gel using amixture of methanol in vacuo (5-30%) and methylene chloride, and/orrecrystallization with methanol or isopropanol. The spectroscopic datafor the coupling products follow.

Compound 4: 2',3',5'-Triacetyl-8-N-morpholine-adenosine carboxyamide ¹ HNMR (DMSO/D₂ O) δ 2.05 (s, 3H), 2.10 (s, 3H), 2.14 (s, 3H), 3.77 (m,2H), 3.86 (m, 6H), 4.36 (m, 2H), 4.49 (m, 1H), 5.84 (t, J=6.3 Hz, 1H),5.92 (s, 2H), 6.12 (dd, J₁ =6.3, J₂ =4.0 Hz, 1H), 6.44 (d, J=4.0 Hz,1H), 8.40 (s, 1H). ¹³ C NMR (DMSO/D₂ O) δ 20.5, 20.5, 20.7, 42.8, 47.9,63.1, 66.6, 66.9, 70.4, 73.3, 79.9, 88.1, 118.3, 142.9, 150.1, 152.2,155.1, 158.7, 169.6, 169.7, 170.6. HRMS (FAB+) m/z 507.1838 (Calc.507.1840 for C₂₁ H₂₆ N₆ O₉ +H⁺).

Compound 5: 2',3',5'-Triacetyl-8-N-(4-methylpyridyl)-adenosinecarboxyamide ¹ H NMR (CDCl₃) δ 2.03 (s, 3H), 2.09 (s, 3H), 2.14 (s, 3H),4.38 (m, 2H), 4.49 (m, 1H), 4.63 (d, J=6.2 Hz, 2H), 5.93 (s, 2H), 5.99(t, J=6.5 Hz, 1H), 6.25 (dd, J₁ =6.4, J₂ =3.6 Hz, 1H), 7.28 (d, J=4.3Hz, 2H), 7.40 (d, J=3.4 Hz, 1H), 8.15 (t, J=6.3 Hz, 1H), 8.36 (s, 1H),8.58 (d, J=4.8 Hz, 2H). ¹³ C NMR (CDCl₃) δ 20.5, 20.7, 42.1, 63.3, 70.4,73.2, 79.6, 88.0, 118.2, 122.2, 140.3, 146.5, 150.1, 151.0, 154.5,156.2, 158.7, 169.6, 169.8, 170.1. HRMS (FAB+) m/z 528.1842 (Calc.528.1843 for C₂₃ H₂₅ N₇ O₈ +H⁺).

Compound 6: 8-N-(4-pyridylmethyl)-adenosine carboxyamide ¹ H NMR (D₂ O)d 3.72 (dd, J₁ =12.5, J₂ =2.3 Hz, 1H), 3.88 (dd, J₁ =10.5, J₂ =1.9 Hz,1H), 4.16 (m, 1H), 4.37 (m, 1H), 4.65 (s, 2H), 4.98 (m, 1H), 7.14 (d,J=7.1 Hz, 1H), 7.43 (d, J=5.6 Hz, 2H), 8.18 (s, 1H), 8.47 (d, J=5.7 Hz,2H). ¹³ C NMR (DMSO/D₂ O) δ 43.0, 64.1, 73.0, 74.7, 88.5, 91.3, 120.1,124.0, 143.2, 150.2, 150.6, 151.5, 154.6, 158.8, 161.0. HRMS (FAB+) m/z402.1522 (Calc. 402.1526 for C₁₇ H₁₉ N₇ O₅ +H⁺).

Compound 7: 8-N-(n-Butyl)-adenosine carboxyamide ¹ H NMR (DMSO) δ 0.91(t, J=7.2 Hz, 3H), 1.34 (m, 2H), 1.52 (m, 2H), 3.31 (t, J=7.2 Hz, 1H),3.53 (m, 1H), 3.68 (m, 2H), 3.95 (d, J=2.7 Hz, 1H), 4.20 (m, 1H), 4.96(dd, J₁ =12.0, J₂ =6.4 Hz, 1H), 5.13 (d, J=4.4 Hz, 1H), 5.25 (d, J=6.4Hz, 1H), 5.59 (dd, J₁ =8.9, J₂ =3.4 Hz, 1H), 6.69 (d, J=6.7 Hz, 1H),7.60 (s, 2H), 8.18 (s, 1H), 8.73 (t, J=5.8 Hz, 1H); ¹³ C NMR (DMSO) δ23.2, 29.1, 40.5, 48.1, 71.8, 80.4, 81.3, 95.8, 98.6, 127.4, 152.2,159.5, 162.9, 166.4, 168.2. HRMS (FAB+) m/z 367.1723 (Calc. 367.1729 forC₁₅ H₂₃ N₆ O₅ +H⁺).

Compound 8: 8-N-(2-Propyl)-adenosine carboxyamide ¹ H NMR (CD₃ OD) δ1.27 (d, J=6.5 Hz, 6H), 3.73 (dd, J₁ =12.5, J₂ =2.6 Hz, 1H), 3.89 (dd,J₁ =12.5, J₂ =2.2 Hz, 1H), 4.17 (m, 1H), 4.21 (q, J=6.5 Hz, 1H), 4.37(dd, J₁ =5.3, J₂ =1.9 Hz, 1H), 4.96 (dd, J₁ =6.9, J₂ =4.3 Hz, 1H), 7.07(d, J=7.2 Hz, 1H), 8.19 (s, 1H). ¹³ C NMR (CD₃ OD) δ 22.5, 43.1, 64.1,72.9, 74.7, 88.4, 91.3, 119.9, 144.0, 151.4, 154.4, 158.7, 159.8. HRMS(FAB) m/z 353.1574 (Calc. 353.1573 for C₁₄ H₂₀ N₆ O₅ +H⁺).

Compound 9: 8-N-(t-Butyl)-adenosine carboxyamide ¹ H NMR (DMSO/D₂ O) δ1.37 (s, 9H), 3.52 (dd, J₁ =12.3, J₂ =3.7 Hz, 1H), 3.66 (dd, J₁ =12.5,J₂ =3.1 Hz, 1H), 3.94 (m, 1H), 4.18 (m, 1H), 4.91 (t, J=5.5 Hz, 1H),6.74 (d, J=6.8 Hz, 1H), 8.15 (s, 1H). ¹³ C NMR (DMSO/D₂ O) δ 28.7, 52.0,62.7, 71.3, 72.4, 86.7, 89.5, 118.1, 143.2, 150.4, 153.9, 157.2, 158.6.HRMS (FAB+) m/z 367.1723 (Calc. 367.1717 for C₁₅ H₂₂ N₆ O₅ +H⁺).

Compound 10: 8-N-Phenyl-adenosine carboxyamide ¹ H NMR (DMSO/D₂ O) δ3.74 (dd, J₁ =12.5, J₂ =2.6 Hz, 1H), 3.91 (dd, J₁ =12.6, J₂ =2.3 Hz,1H), 4.19 (m, 1H), 4.39 (dd, J₁ =5.4, J₂ =2.0 Hz, 1H), 4.99 (dd, J₁=7.1, J₂ =5.5 Hz, 1H), 7.18 (m, 2H), 7.39 (t, J=5.6 Hz, 2H), 7.74 (d,J=5.6 Hz, 2H), 8.21 (s, 1H). ¹³ C NMR (DMSO) δ 62.2, 70.9, 71.9, 86.4,89.1, 118.0, 120.0, 124.5, 128.9, 138.0, 142.7, 150.1, 153.7, 157.0,157.3. HRMS (FAB+) m/z 387.1419 (Calc. 387.1417 for C₁₇ H₁₈ N₆ O₅ +H⁺).

Compound 11: 8-N-(2-(N,N,N'-trimethylammonium)ethyl)-adenosinecarboxyamide chloride. ¹ H NMR (CD₃ OD) δ 3.27 (s, 9H), 3.65 (t, J=6.4Hz, 2H), 3.73 (dd, J₁ =12.5, J₂ =2.5 Hz, 1H), 3.90 (m, 3H), 4.17 (m,1H), 4.36 (dd, J₁ =5.3, J₂ =1.7 Hz, 1H), 4.96 (dd, J₁ =7.1, J₂ =5.4 Hz,1H), 7.16 (d, J=7.2 Hz, 1H), 8.19 (s, 1H). ¹³ C NMR (CD₃ OD) δ 35.0,54.1, 64.1, 65.6, 73.0, 74.6, 88.7, 91.1, 120.1, 142.7, 151.5, 154.7,158.9, 161.1. HRMS (FAB+) m/z 396.1995 (Calc. 396.1995 for C₁₆ H₂₆ N₇ O₅--Cl⁻).

Compound 12: 8-N-(2-hydroxyethyl)-adenosine carboxyamide ¹ H NMR(DMSO/D₂ O) δ 3.35 (m, 2H), 3.53 (m, 3H), 3.67 (dd, J₁ =12.5, J₂ =3.1Hz, 1H), 3.96 (m, 1H), 4.17 (m, 1H), 4.89 (t, J=5.9 Hz, 1H), 6.86 (d,J=6.8 Hz, 1H), 8.15 (s, 1H). ¹³ C NMR (DMSO/D₂ O) δ 42.0, 59.8, 62.7,71.3, 72.4, 86.7, 89.5, 118.0, 142.4, 150.5, 154.0, 157.3159.7. HRMS(FAB) m/z 355.1372 (Calc. 355.1366 for C₁₃ H₁₈ N₆ O₆ +H⁺).

Compound 13: 8-N-(Arginine ethyl ester)-adenosine carboxyamide ¹ H NMR(CD₃ OD) δ 1.28 (t, J=7.1 Hz, 3H), 1.75 (m, 2H), 1.96 (m, 1H), 2.07 (m,1H), 3.27 (m, 2H), 3.73 (dd, J₁ =12.6, J₂ =2.7 Hz, 1H), 3.88 (dd, J₁=12.6, J₂ =2.3 Hz, 1H), 4.18 (m, 1H), 4.23 (q, J=7.1 Hz, 2H), 4.41 (dd,J₁ =5.4, J₂ =1.9 Hz, 1H), 4.67 (dd, J₁ =8.9, J₂ =5.0 Hz, 1H), 5.01 (dd,J₁ =7.0, J₂ =5.5 Hz, 1H), 7.09 (d, J=7.1 Hz, 1H), 8.17 (s, 1H). ¹³ C NMR(D₂ O/CD₃ OD) δ 14.4, 25.6, 28.7, 41.5, 53.8, 63.1, 63.9, 72.0, 74.0,87.5, 90.3, 119.2, 142.4, 150.4, 154.4, 157.4, 157.7, 160.1, 174.0. HRMS(FAB+) m/z (Calc. 387.1417 for C₁₇ H₁₈ N₆ O₅ +H⁺).

Compound 14: 8-N-(t-Butyl)-guanosine carboxyamide ¹ H NMR (CD₃ OD) δ1.44 (s, 9H), 3.74 (dd, J₁ =12.2, J₂ =3.7 Hz, 1H), 3.86 (dd, J₁ =12.2,J₂ =2.9 Hz, 1H), 4.07 (m, 1H), 4.39 (dd, J₁ =5.8, J₂ =3.4 Hz, 1H), 4.95(t, J=6.0 Hz, 1H), 6.93 (d, J=6.4 Hz, 1H). ¹³ C NMR (DMSO/D₂ O) δ 28.9,52.9, 64.0, 72.4, 73.8, 87.4, 91.1, 117.5, 141.6, 154.1, 155.5, 159.7,159.9. HRMS (FAB+) m/z 383.1676 (Calc. 383.1679 for C₁₅ H₂₂ N₆ O₆ +H⁺).

Compound 15: 8-N-(2-(N',N',N'-trimethylammonium)ethyl)guanosinecarboxyamide chloride. ¹ H NMR (D₂ O) δ 3.24 (s, 9H), 3.64 (t, J=6.6 Hz,2H), 3.91 (m, 4H), 4.20 (m, 1H), 4.52 (dd, J₁ =5.1, J₂ =3.7 Hz, 1H),5.05 (t, J=6.0 Hz, 1H), 6.85 (d, J=6.2 Hz, 1H); ¹³ C NMR (CD₃ OD) δ34.6, 54.4, 63.0, 65.0, 71.7, 73.1, 86.6, 90.1, 117.6, 140.1, 153.9,155.1, 160.5, 161.1. HRMS (FAB+) m/z 412.1950 (Calc. 412.1945 for C₁₆H₂₆ N₇ O₆ --Cl⁻).

Compound 16: 8-N-(Arginine ethyl ester)-guanosine carboxyamide ¹ H NMR(CD₃ OD) δ 1.28 (t, J=7.1 Hz, 3H), 1.72 (m, 2H), 1.89 (m, 1H), 2.03 (m,1H), 3.23 (m, 2H), 3.74 (dd, J₁ =12.1, J₂ =3.8 Hz, 1H), 3.86 (dd, J₁=2.2, J₂ =2.9 Hz, 1H), 4.06 (m, 1H), 4.21 (q, J=7.1 Hz, 2H), 4.39 (dd,J₁ =5.7, J₂ =2.4 Hz, 1H), 4.61 (dd, J₁ =9.1, J₂ =4.7 Hz, 1H), 4.98 (t,J=6.0 Hz, 1H), 6.98 (d, J=6.3 Hz, 1H). HRMS (FAB+) m/z 512.2219 (Calc.512.2217 for C₁₉ H₂₉ N₉ O₈ +H⁺).

Compound 17: 8-N-(4-pyridylmethyl)-guanosine carboxyamide ¹ H NMR(DMSO/D₂ O) δ 3.51 (m, 1H), 3.64 (dd, J₁ =11.9, J₂ =4.4 Hz, 1H), 3.79(m, 1H), 4.17 (dd, J₁ =5.5, J₂ =4.3 Hz, 1H), 4.42 (d, J=6.3 Hz, 2H),4.91 (t, J=5.8 Hz, 1H), 6.62 (s, 2H), 6.71 (d, J=5.8 Hz, 1H), 7.29 (d,J=5.7 Hz, 2H), 8.49 (d, J=5.3 Hz, 2H), 9.45 (t, J=6.2 Hz, 1H). ¹³ C NMR(DMSO/D₂ O) δ 41.3, 62.2, 70.4, 71.1, 85.4, 89.0, 116.3, 122.3, 138.3,148.4, 149.6, 152.8, 153.7, 156.7, 159.1. HRMS (FAB+) m/z 418.1482(Calc. 418.1488 for C₁₉ H₂₁ N₄ O₇ +H⁺).

Compound 18: 8-N-(2-Aminoethyl biotinamide)-guanosine carboxyamide ¹ HNMR (CD₃ OD) δ 1.17 (m, 2H), 1.26 (t, J=12.0 Hz, 2H), 1.39 (m, 1H), 1.50(m, 3H), 2.23 (t, J=12.5 Hz, 2H), 2.63 (d, J=22 Hz, 1H), 2.80 (dd, J₁=22, J₂ =8.0 Hz, 1H), 2.90 (m, 1H), 3.18 (m, 2H), 3.48 (m, 4H), 3.81(dd, J₁ =19.5, J₂ =6.0 Hz, 1H), 3.91 (dd, J₁ =11.5, J₂ =4.0 Hz, 1H),4.08 (m, 1H), 4.19 (m, 1H), 4.40 (m, 1H), 4.47 (m, 1H), 4.96 (t, J=10Hz, 1H), 6.83 (d, J=11 Hz, 1H). HRMS (FAB+) m/z 596.2251 (Calc. 596.2264for C₂₃ H₃₃ N₉ O₈ S+H⁺).

EXAMPLE 2 Purine Modification with Alcohols

The general procedure outline in Example 1 was followed to produce themodified purine described by the following scheme with the followingresults. ##STR24## ¹ H NMR (DMSO/D₂ O) δ -0.05 (s, 3H), -0.04 (s, 3H),0.82 (s, 9H), 1.38 (s, 3H), 1.60 (s, 3H), 3.67 (dd, J=10.5, 6.5 Hz, 1H),3.78 (dd, J=10.6, 6.5 Hz, 1H), 4.03 (s, 3H), 4.25 (m, 1H), 5.10 (dd,J=6.5, 3.8 Hz, 1H), 5.69 (dd, J=6.4, 2.1 Hz, 1H), 6.34 (s, 2H), 7.04 (d,J=2.2 Hz, 1H), 8.36 (s, 1H).

EXAMPLE 3 Uridine Modifications with Amines and Alcohols

The following procedures were employed to produce the modified uridinenucleosides described in Table II. ##STR25##

                  TABLE II                                                        ______________________________________                                        Summary of uridine carboxyamidation products.                                         Nucleoside                                                                    starting                   Isolated Yield                             Entry   material  RYH      Product ID                                                                            (%)                                        ______________________________________                                         1      19        A        23      65                                          2      19        B        24      89                                          3      19        C        25      20                                          4      19        D        26      78                                          5      20        E        27      <20                                         6      21        E        28      <20                                         7      20        H        29      69                                          8      21        I        30      68                                          9      21        F        31      61                                         10      21        G        32      80                                         11      21        J        33      68                                         12      21        K        34      57                                         13      21        P        35      43                                         14      21        Q        36      62                                         15      22        B        37      90                                         16      22        L        38      74                                         17      22        M        39      66                                         18      22        N        40      96                                         19      22        O        41      90                                         20      22        A        42      86                                         21      22        P        43      34                                         ______________________________________                                         Starting Material Syntheses.

Compound 22 was prepared according to a literature procedure (P. K.Chang and A. D. Welch, J. Med. Chem. 1963, 6, 428). The other startingmaterials (Compounds 19-21) were synthesized by the followingprocedures.

Compound 19. 5-iodo-2',3'-O-isopropylideneuridine. To a stirred solutionof 5.00 g of 5-iodouridine (13.5 mmol) in 300 mL of acetone was added250 mg of p-toluenesulfonic acid (1.30 mmol). The flask was fitted withan addition funnel filled with 4 Å molecular sieves and a refluxcondenser. The solution was heated at reflux temperature for 2 h., afterwhich all solids had dissolved. The flask was allowed to cool to roomtemperature and the solution concentrated in vacuo. The solution wasdissolved in acetone, filtered through a plug of silica and the filtrateconcentrated to give a pale yellow solid. This material wasre-crystallized from ethanol to give the product as white needles inquantitative yield.

Compound 20. 5'-DMT-5-iodo-2',3'-O-isopropylideneuridine. To a stirredsolution of 820 mg of 5-iodo-2',3'-isopropylideneuridine (2.00 mmol) in1.0 mL of anhydrous DMF and 1.8 mL of anhydrous pyridine, under argon,was added 24.4 mg of 4-dimethylaminopyridine (0.20 mmol) and 745 mg ofDMTCl (2.20 mmol). The solution was stirred at room temperatureovernight, diluted with 150 mL of ethyl acetate, washed with 3×75 mL ofH₂ O, 1×50 mL of brine, and concentrated in vacuo. The residue waspurified on silica gel with 40% EtOAc/hexanes to give 1.28 g (90% yield)of the product as a white solid.

Compound 21. 5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine. To a stirredsolution of 1.00 g of 5-iodo-2',3'-isopropylideneuridine (2.40 mmol) in1.9 mL of anhydrous pyridine was added 724 mg of TBDMSCl (4.80 mmol).The solution was stirred overnight at room temperature, diluted with 30mL of ethyl acetate and washed with 3×20 mL of H₂ O, 1×20 mL of brineand concentrated in vacuo. The residue was purified on silica gel with30% EtOAc/hexanes to give 1.15 g (91% yield) of the product as a whitesolid.

Modified Uridine Syntheses

The modified uridines described in Table II were synthesized as follows.

Compound 23. 5-(N-Butylcarboxyamide)-2',3'-O-isopropylideneuridine. To a300 mL stainless steel Parr bomb in an argon atmosphere glove box wasadded a solution of 5-iodo-2',3'-O-isopropylideneuridine (0.351 g, 1.00mmol, in 3.0 mL of THF), 10 mL of 1.0 M Et₃ N/THF (10 mmol), 3.0 mL of1.0 M n-butylamine in THF (3.0 mmol), andtetrakis(triphenyl-phosphine)palladium (0.116 g, 0.100 mmol). The bombwas sealed, removed from the box, evacuated and charged three times with100 psi CO, then heated at 70° C. for 24 h. The bomb was allowed to coolto room temperature, vented carefully in a fume hood, and the volatilesremoved in vacuo. The crude reaction material was purified on silica gelwith 5% MeOH/CH₂ Cl₂ to give the product as a yellow solid (0.251 g, 65%yield). Analytical samples were obtained by crystallization from MeOH togive the product as fluffy white needles. ¹ H NMR (dmso-d₆) δ 11.92 (brs, 1H), 8.68 (t, J=5.4 Hz, 1H), 8.61 (s, 1H), 5.85 (d, J=1.8 Hz, 1H),5.09 (t, J=4.5 Hz, 1H), 4.91 (dd, J₁ =6.3, J₂ =1.8 Hz, 1H), 4.74 (dd, J₁=6.0, J₂ =2.7 Hz, 1H), 4.19 (m, 1H), 3.56 (m, 2H), 3.24 (m, 2H), 1.47(s, 3H), 1.4 (m, 2H), 1.3 (m, 2H), 1.27 (s, 3H), 0.9 (t, J=7.2 Hz, 3H);¹³ C NMR (dmso-d₆) δ 163.3 (C4), 161.3 (CONHBu), 149.4 (C2), 146.9 (C6),112.5 (CMe₂), 105.0 (C5), 92.8 (C1'), 87.4 (C4'), 84.4 (C2'), 80.7(C3'), 61.2 (C5'), 38.0 (CONHCH₂ --) 31.2, (NHCH₂ CH₂ --), 26.9 (CCH₃),25.0 (CCH₃), 19.5 (NHCH₂ CH₂ CH₂ CH₃), 13.6 (NHCH₂ CH₂ CH₂ CH₃); HRMS:Calculated(observed) for C₁₇ H₂₆ N₃ O₇ : 384.1771(384.1772). Anal.calcd.(found) for C₁₇ H₂₅ N₃ O₇ : C, 53.26(53.46); H, 6.57(6.53); N,10.96(10.98).

Compound 24. 5-N-(4-pyridylmethyl)carboxyamide!-2',3'-O-isopropylideneuridine. To aheavy-walled glass bomb was added 224 mg2',3'-isopropylidene-5-iodouridine (0.542 mmol), 63 mgtetrakis(triphenylphosphine)palladium (0.0542 mmol) and anhydrouspyridine until the solids were dissolved. Pyridine was then removed invacuo and the solids dried under high vacuum overnight. To the bomb wasthen added, under argon, 4 mL anhydrous THF, 0.75 mL triethylamine (5.42mmol) and 0.22 mL 4-aminomethylpyridine (2.17 mmol). The bomb wasevacuated and charged three times with CO and heated to 70° C. for 2.5days. The bomb was allowed to cool to room temperature, the solventremoved in vacuo and the crude material loaded onto a pad of silica withdichloromethane. The pad was eluted with dichloromethane, then thedesired product eluted with 10% MeOH/CH₂ Cl₂ and concentrated in vacuoto a pale yellow solid. This material was purified by flashchromatography on silica gel with 5% MeOH/CH₂ Cl₂ to give 201 mg (89%yield) of the product as a pale yellow solid. This material wasrecrystallized from methanol to give analytical samples of pure productas white needles. ¹ H NMR (dmso-d₆) δ 11.98 (s, 1H), 9.19 (t, J=6.3 Hz,1H), 8.66 (s, 1H), 8.48 (d, 2H, J=4.5 Hz), 7.25 (d, J=5.7 Hz, 2H), 5.86(d, J=2.2 Hz, 1H), 5.10 (t, J=4.8 Hz, 1H), 4.93 (dd, J=6.2, 2.2 Hz, 1H),4.73 (dd, J=6.3, 3.0 Hz, 1H), 4.49 (d, J=6.3 Hz, 2H), 4.20 (m, 1H), 3.56(t, J=4.5 Hz, 2H), 1.47 (s, 3H), 1.27 (s, 3H). ¹³ C NMR (dmso-d₆) δ163.2 (C4), 161.9 (CONH--), 149.5 (pyr o-C), 149.4 (C2), 148.4 (pyrp-C), 147.4 (C6), 122.1 (pyr m-C), 112.6 (CMe₂), 104.8 (C5), 92.9 (C1'),87.4 (C4'), 84.4 (C2'), 80.7 (C3'), 61.2 (C5'), 41.2 (NHCH₂ --), 26.9(CCH₃), 25.0 (CCH₃); HRMS: Calculated(observed) for C₁₉ H₂₃ N₄ O₇ :419.1567(419.1569). UV spectrum: λ_(max) at 276 nm (ε=13730 M⁻¹ cm⁻¹).

5- N-(4-pyridylmethyl)carboxyamide!-5'-triphosphate-uridine. The5'-hydroxyl compound prepared as described was converted to the5'-triphosphate using a modified procedure of Ludwig and Eckstein, J.Org. Chem. 1989, 54, 631-635. After removal of the2',3'-O-isopropylidene protecting group by stirring in H₂ O with DowexH⁺ 50W×80 at 70° C., the crude triphosphate was purified successively onDEAE sephadex anion exchange resin and C18 RP-HPLC using 100 mM Et₃ NH⁺HCO₃ -- and CH₃ CN as the mobile phases. The purity of the compound waschecked by analytical C18 RP-HPLC, ¹ H and ³¹ P NMR (D₂ O), andquantitated by its UV absorbance at 276 nm (ε=13700 M⁻¹ cm⁻¹).

Compound 25. 5-(N-phenylcarboxyamide)-2',3'-O-isopropylideneuridine. Toa heavy-walled glass bomb in an argon atmosphere glove box was added2',3'-isopropylidene-5-iodouridine (0.261 g, 0.636 mmol),tetrakis(triphenylphosphine)palladium (0.083 g, 0.072 mmol), and 4.5 mLof 1.0 M Et₃ N/THF (4.5 mmol). The bomb was sealed, removed from thebox, and 0.3 mL of aniline added via syringe under argon. The flask wasevacuated and charged three times with 50 psi CO and heated to 70° C.for 2 days. The bomb was cooled to room temperature, concentrated invacuo and purified by flash chromatography on silica gel with 4-6.5%MeOH.NH₃ /CH₂ Cl₂ to give a slightly yellow solid. This material wasre-crystrallized from methanol to give 52 mg (20% yield) of the pureproduct as fine white needles. ¹ H NMR (dmso-d₆) δ 12.16 (br s, 1H),10.88 (s, 1H), 8.79 (s, 1H), 7.63 (d, J=7.8 Hz, 2H), 7.34 (m, 2H), 7.09(t, J=7.4 Hz, 1H), 5.88 (d, J=2.1 Hz, 1H), 5.16 (t, J=4.7 Hz, 1H), 4.95(dd, J₁ =6.3, J₂ =2.1 Hz, 1H), 4.76 (dd, J₁ =6.3, J₂ =2.7 Hz, 1H), 4.25(m, 1H), 3.59 (m, 2H), 1.48 (s, 3H), 1.29 (s, 3H); ¹³ C NMR (dmso-d₆) δ163.6 (C4), 159.9 (CONH--), 149.3 (C2), 147.8 (C6), 138.1 (CONHC<),129.0 (phenyl m-C), 124.0 (phenyl p-C), 119.5 (phenyl o-C), 112.5(CMe₂), 104.6 (C5), 93.2 (C1'), 87.6 (C4'), 84.5 (C2'), 80.7 (C3'), 61.2(C5'), 26.9 (CCH₃), 25.0 (CCH₃). HRMS: Calculated(observed) for C₁₉ H₂₂N₃ O₇ : 404.1458(404.1468).

Compound 26. 5'-TBDMS-5-(N-2-(N'-trifluoroacetamido)ethyl!-carboxyamide)-2',3'-O-isopropylideneuridine.To a heavy-walled glass bomb in an argon atmosphere glove box was added5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine (0.531 g, 1.01 mmol),tetrakis(triphenylphosphine)palladium (0.350 g, 0.303 mmol), Et₃ N(0.704 mL, 5.05 mmol) and 2 mL of dry THF. The bomb was sealed, removedfrom the box and 0.203 mL ethylenediamine (3.03 mmol) added underpositive argon flow. The bomb was sealed under argon, evacuated andcharged three times with 50 psi CO and heated to 70° C. overnight. Thebomb was allowed to cool to room temperature, vented slowly, the solventremoved in vacuo and the crude material purified on silica gel with 25%MeOH.NH₃ /EtOAc to give 381 mg (78% yield) of the product as a whitesolid. This material was protected as the N-triflouroacetamide in thefollowing manner. To a stirred solution of 381.0 mg of the above product(0.78 mmol) in 7.0 mL of anhyd. CH₂ Cl₂ at 0° C. was added dry pyridine(0.126 mL, 1.6 mmol) and (CF₃ CO)₂ O (0.13 mL, 0.94 mmol). The solutionwas stirred at 0° C. for 30 min. then 0.19 mL of (CF₃ CO)₂ O (1.33 mmol)and 0.13 mL of pyridine (1.7 mmol) was added. After 30 min. the reactionwas allowed to warm to room temperature, concentrated in vacuo andpurified by flash silica gel chromatography with 40% EtOAc/hexanes togive 174 mg (38% yield, 30% yield from iodouridine starting material) ofthe product as a white solid. ¹ H NMR (dmso-d₆) δ 11.95 (s, 1H), 9.48(t, J=5.0 Hz, 1H), 8.81 (t, J=5.8 Hz, 1H), 8.49 (s, 1H), 5.75 (d, J=1.6Hz, 1H), 4.89 (dd, J₁ =6.1, J₂ =1.7 Hz, 1H), 4.67 (dd, J₁ =6.1, J₂ =2.2Hz, 1H), 4.36 (m, 1H), 3.77 (m, 2H), 3.4 (m, 2H), 3.3 (m, 2H), 1.48 (s,3H), 1.29 (s, 3H), 0.78 (s, 9H), 0.00 (s, 3H), -0.04 (s, 3H); ¹³ C NMR(dmso-d₆) δ 163.1 (C4), 162.0 (CONH--), 149.4 (C2), 147.0 (C5), 117.7,113.9, 112.2, 104.4, 94.7, 87.8, 85.0, 81.0, 63.4, 38.1, 37.2, 26.8,25.6, 24.9, 17.8, -5.8, -5.8. HRMS: Calculated(observed) for C₂₃ H₃₆ F₃N₄ O₈ Si: 581.2254(581.2249).

5'-Triphosphate-5- N-(2-aminoethyl)carboxyamide!-uridine. The 5'-TBDMSprotected ethylenediamine amide of uridine (prepared above) wasdesilylated with Et₃ NH⁺ F⁻ in CH₃ CN for 2 days and purified on silicagel with 20% MeOH/CH₂ Cl₂ to give the 5'-hydroxyl compound, asidentified by ¹ H and ¹³ C NMR, and FAB⁺ mass spectrometry. Thiscompound was used for the preparation of the 5'-triphosphate using amodified of procedure of Ludwig and Eckstein, J. Org. Chem. 1989, 54,631-635. After removal of the 2',3'-O-isopropylidene protecting group bystirring in H₂ O with Dowex H⁺ 50W×80 at 70° C., the product waspurified on C18 RP-HPLC with 0.05 M TBK/CH₃ CN as the mobile phase togive desired product in 9% yield. The product was characterized by ¹ Hand ³¹ P NMR and FAB⁺ MS.

Compound 27. 5'-DMT-5-carbomethoxy-2',3'-O-isopropylideneuridine. In aglove box 5'-DMT-5-iodo-2',3'-O-isopropylideneuridine (1.0 mL of asolution of 0.10 g/mL, 0.14 mmol) was added to a small heavy-walledglass bomb. Solid tetrakis(triphenylphosphine)-palladium (16 mg, 0.014mmol) was added, followed by 0.70 mmol of Et₃ N as a 1.0 M solution inTHF, and 3.0 mL of anhydrous methanol (distilled in vacuo over Mg). Thebomb was evacuated and refilled with 50 psi of CO (3×), then sealed andheated to 70° C. with stirring for 3 days. The vessel was vented and thesolvents removed in vacuo, and the residue dissolved in the minimum 5%MeOH/CH₂ Cl₂, loaded onto a pad of silica gel, and eluted successivelywith CH₂ Cl₂ (discarded) and 5% MeOH/CH₂ Cl₂. The resultant material waspurified on silica gel with 5% MeOH/CH₂ Cl₂ to give the product as acolorless solid. ¹ H NMR (CD₃ OD) δ 8.67 (s, 1H), 7.6-6.8 (m, 13H), 5.83(d, J=2.1 Hz, 1H), 4.98 (dd, J₁ =6.2, J₂ =2.1 Hz, 1H), 4.59 (m, 1H),4.32 (m, 1H), 3.76 (two s, total 6H), 3.41 (s, 3H), 3.36 (m, 2H), 1.51(s, 3H), 1.29 (s, 3H). FAB⁺ m/z 667 (M+Na⁺), 645 (M+H⁺), 303 (DMT⁺).

Compound 28. 5'-TBDMS-5-carbomethoxy-2',3'-O-isopropylideneuridine. Thiscompound was prepared as described above for the 5'-DMT protectedcompound, except using 5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine asthe starting material. The product was isolated by flash chromatographyon silica gel as a colorless solid. ¹ H NMR (CD₃ OD) δ 8.58 (s, 1H),5.71 (d, J=2.1 Hz, 1H)), 4.89 (dd, J₁ =6.2, J₂ =2.2, 1H), 4.75 (dd, J₁=6.0, J₂ =1.8 Hz), 4.49 (m, 1H), 3.88 (m, 2H), 3.78 (s, 3H), 1.53 (s,3H), 1.34 (s, 3H), 0.83 (s, 9H), 0.05 (s, 3H), -0.01 (s, 3H). ¹³ C NMR(CD₃ OD) δ 164.9 (C4), 162.2 (COOMe), 151.3 (C2), 150.3 (C6), 114.4(CMe₂), 104.4 (C5), 97.3 (C1'), 90.1 (C4'), 87.4 (C2'), 83.1 (C3'), 65.1(C5'), 52.4 (OCH₃), 27.4 (CCH₃), 26.3 (SiC CH₃ !₃), 25.3 (CCH₃), 19.2(SiC CH₃ !₃), -5.4 (SiCH₃), -5.5 (SiCH₃). HRMS: Calculated(observed)mass for C₂₀ H₃₃ N₂ O₈ Si: 457.2006(457.2006).

Compound 29.5'-TBDMS-5-(N-histidinolcarboxyamide)-2',3'-O-isopropylideneuridine. Toa heavy-walled glass bomb in an argon atmosphere glove box was added 3.5mL of a 100 mg/mL solution of5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine (0.491 mmol), 57 mgtetrakis(triphenylphosphine)palladium (0.0491 mmol), 0.2 mL oftriethylamine (1.473 mmol) and 0.5 mL of THF. The bomb was sealed,removed from the box and under argon 1.9 mL of a 100 mg/mL solution ofTBDMS protected histidinol (0.736 mmol) was added. The bomb was sealedunder argon, evacuated and charged three times with 50 psi CO, andheated at 70° C. for 48 h. The bomb was allowed to cool to roomtemperature, vented and the solvent removed in vacuo. The crude materialwas purified by chromatography on silica gel with either a gradient of5-7% or 0-5% MeOH/CH₂ Cl₂ to give 0.294 g (69% yield) of the desiredproduct as a white solid. ¹ H NMR (dmso-d₆) δ 11.9 (br s, 2H), 8.9 (d,J=8.3 Hz, 1H), 8.6 (s, 1H), 7.5 (s, 1H), 7.3 (m, 9H), 6.8 (m, 4H), 6.7(s, 1H), 5.9 (d, J=1.3 Hz, 1H), 5.0 (dd, J₁ =6.3, J₂ =1.4 Hz, 1H), 4.5(unres. dd, 1H), 4.2 (m, 2H), 3.6 (m, 2H), 3.3 (m, 2H), 2.5 (m, 2H), 1.5(s, 3H), 1.2 (s, 3H), 0.9 (s, 9H), 0.01 (s, 6H). ¹³ C NMR (dmso-d₆) δ163.2, 160.8, 158.0, 149.2, 148.0, 144.7, 135.3, 135.2, 134.7, 129.7,129.5, 127.7, 127.5, 126.6, 113.1, 112.9, 105.2, 93.8, 86.4, 85.7, 83.9,80.7, 63.9, 63.1, 54.9, 50.1, 28.5, 26.8, 25.7, 25.0, 17.9, -5.6, -5.6.HRMS: Calculated(observed) mass for C₄₆ H₅₇ N₅ O₁₀ Si:867.3874(867.3884).

Compound 30. 5'-TBDMS-5- N-(2-4-imidazole!ethyl)carboxyamide!-2',3'-O-isopropylideneuridine. To aheavy-walled glass bomb in an argon atmosphere glove box was added5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine (0.260 g, 0.496 mmol), 4mL of dry THF, and tetrakis(triphenylphosphine)palladium (0.073 g, 0.063mmol). The bomb was sealed, removed from the box and the solvent removedin vacuo. Under argon, anhydrous Et₃ N (0.35 mL, 2.48 mmol), histamine(0.263 g, 2.37 mmol) and 2 mL of dmso-d₆ were added. The bomb wasevacuated and charged three times with 50 psi CO and heated at 70° C.for 2 days. After cooling to room temperature, the bomb was ventedcarefully and the solvents removed in vacuo at 70° C. The crude materialwas purified by flash chromatography on silica gel with 12% MeOH/CH₂ Cl₂to give 181 mg (68% yield) as a slightly yellow solid. ¹ H NMR (CD₃ OD)δ 8.6 (s, 1H), 7.6 (s, 1H), 6.9 (s, 1H), 5.7 (d, J=1.9 Hz, 1H), 4.7 (dd,J₁ =5.9, J₂ =1.6 Hz, 1H), 4.5 (m, 1H), 3.9 (m, 2H), 3.6 (m, 2H), 2.8 (t,2H), 1.5 (s, 1H), 1.3 (s, 1H), 0.8 (s, 9H), 0.04 (s, 3H), -0.01 (s, 3H);¹³ C NMR (dmso-d₆) δ 163.2, 161.3, 149.4, 146.7, 134.7, 112.2, 111.8,104.6, 94.6. 87.8, 84.9, 81.0, 63.4, 48.5, 27.0, 26.8, 25.6, 24.9, 17.9,-5.7; HRMS: Calculated(observed) for C₂₄ H₃₇ N₅ O₇ Si:535.2462(535.2456). UV spectrum: λ_(max) at 278 nm (ε=12930 M⁻¹ cm⁻¹).

5'-Triphosphate-5- N-(2- 4-imidazole!ethyl)carboxyamide!-uridine. The5'-TBDMS protected histamine amide of uridine (prepared above) wasdesilylated with Et₃ NH⁺ F⁻ in CH₃ CN for 2 days and purified on silicagel with 15% NH₃ -MeOH/CH₂ Cl₂ to give the 5'-hydroxyl histamine amideof uridine, as identified by ¹ H and ¹³ C NMR, and FAB⁺ massspectrometry. This compound was used for the preparation of the5'-triphosphate using a modified procedure of Ludwig and Eckstein, J.Org. Chem. 1989, 54, 631-635. After removal of the2',3'-O-isopropylidene protecting group with acidic Dowex resin in H₂ Oat 70° C., the crude triphosphate was purified successively on DEAEsephadex anion exchange resin and C18 RP-HPLC using 100 mM aq. Et₃ NH⁺HCO₃ -- and CH₃ CN as the mobile phases. The purity of the compound waschecked by analytical C18 RP-HPLC, ¹ H and ³¹ P NMR (D₂ O), andquantitated by its UV absorbance at 278 nm (using the ε for thenucleoside starting material, ε₂₇₈ =12930 M⁻¹ cm⁻¹).

Compound 31. 5'-TBDMS-5-N-(2-hydroxyethyl)carboxyamide!-2',3'-O-isopropylideneuridine. Thiscompound was prepared as described above for compound 30, using 3 eq. ofethanolamine and 3 eq. of triethylamine, and allowed to react for 48 hat 70° C. The product was purified on silica gel with 6% MeOH/CH₂ Cl₂ togive 0.173 g (61% yield) of colorless white solid. ¹ H NMR (dmso-d₆) δ11.93 (s, 1H), 8.80 (t, J=5.6 Hz, 1H), 8.48 (s, 1H), 5.75 (d, J=1.8 Hz,1H), 4.89 (dd, J₁ =6.1, J₂ =1.8 Hz, 1H), 4.78 (t, J=5.1 Hz, 1H), 4.67(unres. dd, 1H), 4.34 (m, 1H), 3.76 (d, J=3.8 Hz, 2H), 3.44 (m, 2H),3.31 (m, 2H), 1.47 (s, 3H), 1.28 (s, 3H), 0.78 (2, 9H), -0.02 (s, 3H),-0.04 (s, 3H). ¹³ C NMR (dmso-d₆) δ 163.2, 161.5, 149.4, 146.8, 112.3,104.6, 94.5, 87.7, 84.9, 80.9, 63.4, 59.7, 41.2, 26.8, 25.6, 24.9, 17.9,-5.7, -5.8. Analytical sample from EtOAc/Hexanes. FAB⁺ HRMScalculated(observed) for C₂₁ H₃₆ N₃ O₈ Si: 486.2272(486.2271). Anal.Calcd. (Found) for C₂₁ H₃₅ N₃ O₈ Si: C, 51.94 (52.03); H, 7.26 (7.36);N, 8.65 (8.61).

Compound 32. 5'-TBDMS-5-N-((2-carboethoxy)ethyl)carboxyamide!-2',3'-O-isopropylideneuridine.This compound was prepared as described above for compound 30, using 1.0eq. of glycine ethyl ester hydrochloride and 3 eq. of triethylamine. Theproduct was purified on silica gel with 4% MeOH/CH₂ Cl₂ to give 0.262 g(80% yield) of colorless white solid. ¹ H NMR (CDCl₃) δ 8.96 (t, J=5.5Hz, 1H), 8.72 (s, 1H), 8.65 (s, 1H), 5.74 (d, J=2.2 Hz, 1H), 4.85 (dd,J₁ =6.0, J₂ =2.2 Hz, 1H), 4.72 (dd, J₁ =6.0, J₂ =1.6 Hz, 1H), 4.51 (m,1H), 4.22 (q, J=7.1, 2H), 4.14 (d, J=5.6 Hz, 2H), 3.96 (m, 1H), 3.78 (m,1H), 1.58 (s, 3H), 1.36 (s, 3H), 1.28 (t, J=7.1 Hz, 3H), 0.82 (s, 9H),0.04 (s, 3H), -0.01 (s, 3H). ¹³ C NMR (CDCl₃) δ 169.6, 163.1, 161.8,149.4, 147.3, 112.3, 104.1, 94.6, 87.8, 84.9, 81.0, 63.4, 60.4, 40.8,26.9, 25.6, 24.9, 17.9, 14.0, -5.8. Analytical sample fromEtOAc/Hexanes. FAB⁺ HRMS calculated(observed) for C₂₃ H₃₈ N₃ O₉ Si:528.2377(528.2382). Anal. Calcd. (Found) for C₂₃ H₃₇ N₃ O₉ Si: C, 52.36(52.19); H, 7.07 (6.93); N, 7.96 (7.85).

Compound 33. 5'-TBDMS-5-morpholinecarboxyamide!-2',3'-O-isopropylideneuridine. This compound wasprepared as described above for compound 30, using 3 eq. of morpholineand 3 eq. of triethylamine. The product was purified on silica gel with4% MeOH/CH₂ Cl₂ 0.202 g (68% yield) of colorless white solid. ¹ H NMR(dmso-d₆) δ 11.65 (s, 1H), 7.90 (s, 1H), 5.80 (d, J=2.2 Hz, 1H), 4.93(dd, J=6.2, 2.2 Hz, 1H), 4.68 (6.2, 3.4 Hz, 1H), 4.12 (m, 1H), 3.76 (m,2H), 3.5 (br m, 6H), 3.29 (br m, 2H), 1.47 (s, 3H), 1.28 (s, 3H), 0.84(s, 9H), 0.03 (s, 6H). ¹³ C NMR (dmso-d₆) δ 162.3, 160.3, 149.7, 142.2,112.9, 111.1, 66.2, 65.9, 63.0, 47.0, 41.9, 26.9, 25.8, 25.1, 18.0,-5.5, -5.6. Analytical sample from EtOAc/Hexanes. FAB⁺ HRMScalculated(observed) for C₂₃ H₃₈ N₃ O₈ Si: 512.2428(512.2436).

Compound 34. 5'-TBDMS-5- N-(arginine ethylester)carboxyamide!-2',3'-O-isopropylideneuridine. To a heavy-walledglass bomb in an argon atmosphere glove box was added5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine (0.238 g, 0.453 mmol),arginine ethyl ester dihydrochloride (0.260 g, 0.94 mmol),tetrakis(triphenylphosphine)-palladium (0.052 g, 0.045 mmol), Et₃ N(0.32 mL, 2.3 mmol), 3 mL of dry THF, and 2 mL of DMSO. The reactionvessel was evacuated and charged with 50 psi of CO three times, thenheated to 70° C. for 2 days. The crude mixture was concentrated andchromatographed on silica gel with 25% MeOH--NH₃ /EtOAc to give theproduct as an off-white solid, 0.160 g (57% yield). ¹ H NMR (CD₃ OD) δ8.55 (s, 1H), 5.64 (d, J=1.9 Hz, 1H), 4.77 (unres. dd, 1H), 4.68 (unresdd, 1H), 4.22 (q, J=7.1 Hz, 2H), 4.17 (m, 1H), 3.93 (m, 1H), 3.75 (m,2H), 3.43 (br m, 2H), 2.0 (br m, 1H), 1.78 (br m, 3H), 1.50 (s, 3H),1.34 (s, 3H), 1.26 (t, J=7.1 Hz, 3H), 0.92 (s, 9H), 0.13 (s, 6H).

Compound 35. 5- N-(2-3-indolyl!ethyl)carboxyamide)-2',3'-O-isopropylideneuridine. To aheavy-walled glass bomb in an argon atmosphere glove box was added5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine (1.13 g, 2.14 mmol),2-(3-indolyl)ethylamine (1.70 g, 10.7 mmol) andtetrakis(triphenylphosphine)palladium (0.247 g, 0.214 mmol), anhydroustriethylamine (1.5 mL, 10.7 mmol), and 10 mL of THF. The bomb wassealed, and evacuated and charged three times with 50 psi CO, thenheated at 70° C. for 16 h. After cooling, and removal of the solvents invacuo, the crude material was purified on silica gel with 0-5% MeOH/CH₂Cl₂ to give 0.895 g of slightly impure yellow solid (78% crude yield).Desilylation was accomplished by stirring the above material in 2 mL ofanhyd. CH₃ CN with Et₃ N.HF (1.0 g, 8.4 mmol) for 16 h at ambient temp.The reaction mixture was diluted with 30 mL of EtOAc and extracted with3×20 mL of H₂ O, 10 mL of brine, then concentrated in vacuo and purifiedon silica gel with 0-5% MeOH/CH₂ Cl₂ to give the desired product as apale yellow solid (0.430 g, 43% yield). ¹ H MNR (CD₃ OD) δ 10.2 (br s,1H), 9.1 (t, 1H), 8.7 (s, 1H), 7.5 (d, J=7.8 Hz, 1H), 7.3 (d, J=8.1 Hz,1H), 7.0 (m, 3H), 5.9 (d, J=2.1 Hz, 1H), 4.3 (t, 1H), 3.7 (m, 4H), 3.0(t, 2H), 1.5 (s, 1H), 1.3 (s, 1H).

5'-Triphosphate-5- N-(2- 3-indolyl!ethyl)carboxyamide)uridine. The5'-TBDMS protected tryptamine amide of uridine (prepared above) wasdesilylated with 5 eq. of Et₃ NH⁺ F⁻ in CH₃ CN for 18 h at RT andpurified on silica gel to give the 5'-hydroxyl compound in 43% yield, asidentified by ¹ H and ¹³ C NMR, and FAB⁺ mass spectrometry. The5'-hydroxyl compound was then used for the preparation of the5'-triphosphate using a modified of procedure of Ludwig and Eckstein, J.Org. Chem. 1989, 54, 631-635. After removal of the2',3'-O-isopropylidene protecting group by stirring in H₂ O with DowexH⁺ 50W×80 at 70° C., the product was purified on DEAE sephadex with0.05-1.5 M TBK buffer with 25% added CH₃ CN, followed by C18 RP-HPLCwith 0.05 M TBK/CH₃ CN mobile phase as eluant. The triphosphate wascharacterized by ¹ H and ³¹ P NMR and FAB⁺ MS.

Compound 36. 5'-TBDMS-5-(N-6-aminohexyl!-carboxyamide)-2',3'-O-isopropylideneuridine. To aheavy-walled glass bomb in an argon atmosphere glove box was added5'-TBDMS-5-iodo-2',3'-O-isopropylideneuridine (1.28 g, 2.40 mmol),1,6-diaminohexane (1.40 g, 12.0 mmol) andtetrakis(triphenylphosphine)palladium (0.83 g, 0.72 mmol) and 10 mL ofTHF. The bomb was sealed, removed from the box and triethylamine (1.7mL, 12.0 mmol) was added under argon via syringe. The vessel wasevacuated and charged three times with 50 psi CO and heated at 70° C.overnight. The bomb was allowed to cool, vented, and the solvent removedin vacuo. The crude material was dissolved in 10 mL of methanol and thepalladium catalyst removed by filtration. The filtrate was concentratedand purified on silica gel with 15-25% NH₃ --CH₃ OH in CH₂ Cl₂ to givethe desired product (0.812 g, 62% yield) as a white solid. ¹ H NMR (CD₃OD) δ 9.0 (s, 1H), 5.7 (d, J=1.8 Hz, 1H), 4.9 (m, 1H), 4.7 (m, 1H), 4.5(br s, 1H), 3.9 (m, 2H), 3.3 (m, 3H), 2.9 (t, 2H), 1.5 (unres. m, 12H),0.8 (s, 9H), 0.04 (s, 3H), 0.0 (s, 3H).

5-(N-6-N'-trifluoroacetamidohexyl!-carboxyamide)-2',3'-O-isopropylideneuridine.The free amine of the above compound was protected in the followingmanner. To a stirred solution of compound 36 in acetonitrile withcatalytic triethylamine was added 2.0 eq. ethyl triflouroacetate. Thesolution was stirred at RT for 18 h, and an additional 1.6 eq. ethyltriflouroacetate added and the solution stirred for 20 h. Concentrationand purification on silica gel with 1-5% MeOH/CH₂ Cl₂ gave the desiredtriflouroacetamide in 25% yield, as characterized by ¹ H NMR. Thecompound was then desilylated with 5 eq. of Et₃ NH⁺ F⁻ in CH₃ CN for 18h at RT and purified on silica gel to give the 5'-hydroxyl compound inquantitative yield, as identified by its ¹ H NMR spectrum.

General prodedure for compounds 37-43. To a glass pressure reactorequipped with a Teflon valve were added nucleotide (0.1 mmol), the amine(0.5 mmol, 5 eq.), Pd(PPh₃)₄ (0.01 mmol, 0.1 eq.), and dimethylsulfoxide as solvent (0.5 mL). The reactor was evacuated and chargedwith CO (50 psi) three times before heating to 60° C. for 24 hours. Thecrude mixture was purified on a DEAE Sephadex A-25 anion exchange columnusing a linear gradient of 0.05 M to 1.0 M triethylammonium bicarbonatebuffer. Characterization of the compounds follows.

Compound 37. ¹ H NMR (D₂ O) δ 4.0 (m, 2H), 4.23 (m, 1H), 4.28 (t, J=4.5Hz, 1H), 4.43 (t, J=5.2 Hz, 1H), 4.60 (s, 2H), 5.93 (d, J=5.1 Hz, 1H),7.34 (d, J=4.8 Hz, 2H), 8.42 (d, J=4.8 Hz, 2H), 8.55 (s, 1H). ¹³ C NMR(CD₃ OD) δ 43.2, 64.9, 71.1, 74.9, 85.1, 90.7, 106.9, 123.4, 147.7,149.4, 150.2, 152.1, 165.0, 165.5. ³¹ P NMR (CD₃ OD) δ 6.4. HRMS (FAB+)m/z 459.0909 (Calc. 459.0917 for C₁₆ H₁₉ N₄ O₁₀ P+H⁺).

Compound 38. ¹ H NMR (D₂ O) δ 1.79 (m, 2H), 2.20 (t, J=7.5 Hz, 2H), 3.34(t, J=6.9 Hz, 2H), 4.08 (m, 2H), 4.28 (m, 2H), 4.37 (t, J=4.8 Hz, 1H),5.92 (d, J=4.9 Hz, 1H), 8.55 (s, 1H). ¹³ C NMR (D₂ O) δ 26.8, 36.0,39.9, 65.0, 71.3, 74.6, 84.4, 90.6, 107.2, 146.2, 159.3, 167.1, 174.4,183.6. ³¹ P NMR (CD₃ OD) δ 8.2. MS (FAB) m/z 454.0858 (Calc. 454.0863for C₁₄ H₂₀ N₃ O₁₂ P+H⁺).

Compound 39. ¹ H NMR (D₂ O) δ 2.73 (t, J=6.7 Hz, 2H), 3.50 (t, J=6.7 Hz,2H), 4.03 (m, 2H), 4.25 (m, 2H), 4.36 (t, J=4.8 Hz, 1H), 5.87 (d, J=4.9Hz, 1H), 6.76 (d, J=8.3 Hz, 2H), 7.08 (d, J=8.3 Hz, 2H), 8.44 (s, 1H).¹³ C NMR (CD₃ OD) δ 34.6, 41.9, 65.0, 71.0, 74.9, 8439, 90.7, 106.9,116.4, 131.2, 131.9, 147.2, 151.8, 155.0, 164.6, 164.7. ³¹ P NMR (CD₃OD) δ 8.1. HRMS (FAB+) m/z 488.1082 (Calc. 488.1084 for C₁₈ H₂₂ N₃ O₁₁P+H⁺).

Compound 40. ¹ H NMR (D₂ O) δ 0.87 (d, J=6.7 Hz, 6H), 1.82 (m, 1H), 4.03(m, 2H), 4.27 (m, 2H), 4.39 (t, J=5.0 Hz, 1H), 5.91 (d, J=5.1 Hz, 1H),8.53 (s, 1H). ¹³ C NMR (MeOD) δ 20.3, 29.0, 43.3, 65.1, 71.0, 75.0,85.0, 90.6, 107.2, 147.3, 151.9, 164.8, 165.0. ³¹ P NMR (CD₃ OD) δ 6.4.HRMS (FAB+) m/z 424.1129 (Calc. 424.1121 for C₁₄ H₂₂ N₃ O₁₀ P+H⁺).

Compound 41. ¹ H NMR (D₂ O) δ 1.16 (d, J=5.4 Hz, 6H), 4.02 (m, 3H), 4.28(m, 2H), 4.38 (t, J=5.0 Hz, 1H), 5.90 (d, J=3.6 Hz, 1H), 8.50 (s, 1H).¹³ C NMR (D₂ O) δ 164.9, 163.7, 151.9, 147.1, 107.2, 90.6, 85.0, 75.0,71.0, 65.1, 43.0, 22.5. ³¹ P NMR (D₂ O) δ 6.5.

Compound 42. ¹ H NMR (D₂ O) δ 0.86 (t, J=7.3 Hz, 3H), 1.32 (m, 2H), 1.51(m, 2H), 3.31 (m, 2H), 3.99 (m, 2H), 4.23 (m, 1H), 4.28 (m, 1H), 4.40(t, J=5.1 Hz, 1H), 5.91 (d, J=5.1 Hz, 1H), 8.50 (s, 1H). ¹³ C NMR (1:1D₂ O:CD₃ OD) δ 14.0, 20.7, 31.8, 40.0, 65.0, 71.4, 75.0, 85.3, 90.5,107.0, 147.2, 152.8, 164.8, 166.1. ³¹ P NMR (D₂ O) δ 7.7. HRMS (FAB+)m/z 424.1121 (Calc. 424.1121 for C₁₄ H₂₂ N₃ O₁₀ P+H⁺).

Compound 43. H NMR (D₂ O) δ 2.88 (t, J=6.3 Hz, 2H), 3.53 (t, J=6.3 Hz,2H), 3.96 (s, 2H), 4.20 (d, J=2.3 Hz, 2H), 4.30 (t, J=4.0 Hz, 1H), 5.78(d, J=4.7 Hz, 1H), 6.97 (t, J=7.5 Hz, 1H), 7.08 (m, 2H), 7.34 (d, J=8.0Hz, 1H), 7.46 (d, J=7.8 Hz, 1H), 8.22 (s, 1H). ¹³ C NMR (D₂ O) δ 25.0,40.1, 64.8, 71.1, 74.7, 84.8, 90.8, 106.8, 112.7, 119.5, 120.0, 122.8,124.5, 127.8, 137.2, 146.6, 152.7, 164.8, 165.9. ³¹ P NMR (MeOD) δ 8.2.HRMS (FAB+) m/z 511.1236 (Calc. 511.1230 for C₂₀ H₂₃ N₄ O₁₀ P+H⁺).

EXAMPLE 4 Cytidine Modifications with Amines

The following procedures were employed to produce the modified cytidinesshown in Table III. ##STR26##

                  TABLE III                                                       ______________________________________                                        Summary of cytidine carboxyamidation products.                                          Starting                Isolated Yield                              Entry     material                                                                              RNH.sub.2                                                                             Product ID                                                                            (%)                                         ______________________________________                                        1.sup.a   44      A       45      73                                          2.sup.b   44      B       46      42                                          3.sup.a   44      C       47      80                                          4.sup.c   44      D       48      36                                          5.sup.c   44      E       49      20                                          6.sup.b   44      F       50      49                                          6.sup.c   44      G       51      81                                          ______________________________________                                         .sup.a Nucleotide (0.1 mmol), RNH.sub.2 (0.5 mmol), Pd(PPh.sub.3).sub.4       (0.01 mmol), DBU (0.5 mmool), DMSO (0.5 mL).                                  .sup.b Nucleotide (0.1 mmol), RNH.sub.2 (0.5 mmol), Pd.sub.2 (dba).sub.3      (2.5 μmol), P(pC.sub.6 H.sub.4 --SO.sub.3 Na).sub.3 (0.015 mmol), DABC     (0.7 mmol), DMSO (0.5 mL).                                                    .sup.c Nucleotide (0.1 mmol), RNH.sub.2 (0.5 mmol), Pd(PPh.sub.3).sub.4       (0.01 mmol), DBU or DABCO (0.5 mmol), DMSO:H.sub.2 O 93:7 (0.5 mL).           General Information

5-Iodocytidine monophosphate was synthesized according to a literatureprocedure (Voytek, P.; Chang, P. K.; Prusoff, W. H. J. Biol. Chem. 1971,246, 1432). Trisulfonated triphenylphosphine sodium salt was purchasedfrom Strem Chemicals, Inc. All other compounds were purchased fromAldrich Chemical Co. n-Butylamine, isopropylamine, triethylamine, and1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) were purchased from AldrichChemical Co. and distilled from CaH₂. Isobutylamine, tyraminehydrochloride, and 4-aminobutyric acid (Acros Organics) were used as is.Histamine was purchased from Sigma Chemical Co. and used as is. Cytidinemonophosphate, Pd₂ (dba)₃, tetrakis(triphenylphosphine)palladium(0),DABCO, 4-aminomethylpyridine, and DMSO were purchased from AldrichChemical Co. and used as is. The ¹ H and ¹³ C NMR spectra were obtainedin CD₃ OD, D₂ O, or DMSO-d⁶ using a Bruker ARX-300 spectrometer andreferenced to solvent resonances. High resolution fast atom bomdardmentmass spectra (HR FAB MS) were obtained using VG 70 SE & ZAB2-EQ/FAB(+).

General procedure

The general procedure to synthesize the compounds of Table III isprovided. To a heavy-walled glass pressure reactor equipped with aTeflon valve were added nucleotide (0.1 mmol), amine (0.5 mmol),Pd(PPh₃)₄ (0.01 mmol), DBU (0.5 mmol), DMSO (0.5 ml). The reactor wasevacuated and charged with CO (50 psi) three times before heating to 60°C. for 24 hours. The crude mixture was quantitatively analyzed byreverse phase HPLC. The products were purified on a DEAE Sephadex A-25ion exchange column using a linear gradient (0.05 M to 1.0 M) oftriethylammonium bicarbonate followed by preparative C-18 reverse phaseHPLC (0.05 M thiethylammonium hydrogen carbonate/MeOH). Characterizationof the compounds listed in Table III follows.

Compound 45. 5-(N-Butylcarboxyamide)-cytidine monophosphate. ¹ H NMR(CD₃ OD) δ 0.85 (t, J=7.4 Hz, 3H), 1.29 (m, 2H), 1.49 (m, 2H), 3.24 (m,2H), 4.05 (m, 2H), 4.25 (m, 2H), 4.33 (m, 1H), 5.87 (d, J=4.1 Hz, 1H),8.38 (s, 1H). ¹³ C NMR (CD₃ OD) δ 14.2, 20.7, 31.7, 40.7, 64.6, 70.8,75.6, 84.9. 91.1, 102.8, 144.3, 157.3, 164.8, 167.0. ³¹ P NMR (CD₃ OD) δ5.8. HRMS (FAB+) m/z 423.1275 (Calc. 423.1281 for C₁₄ H₂₃ N₄ O₉ P+H⁺).

Compound 46. 5-(N-Isobutylcarboxyamide)-cytidine monophosphate. ¹ H NMR(CD₃ OD) δ 0.92 (d, J=3.0 Hz, 3H), 0.94 (d, J=3.0 Hz, 3H), 1.99 (m, 1H),3.12 (m, 2H, overlapped with Et₃ NH⁺), 4.07 (m, 1H), 4.16 (m, 2H), 4.29(m, 2H), 5.99 (d, J=4.9 Hz, 1H), 8.66 (s, 1H). ¹³ C NMR (60:1 D₂ O:CD₃OD) δ 20.6, 20.6, 29.0, 48.2, 64.8, 70.7, 75.5, 84.6, 91.4, 103.0,144.3, 157.3, 164.8, 167.3.

Compound 47. 5-(N-Isopropylcarboxyamide)-cytidine monophosphate. ¹ H NMR(CD₃ OD) δ 1.22 (m, 6H), 4.06 (m, 3H), 4.24 (m, 1H), 4.28 (m, J=4.9 Hz,1H), 4.38 (t, J=5.0 Hz, 1H), 5.89 (d, J=4.8 Hz, 1H), 8.31 (s, 1H). ¹³ CNMR (CD₃ OD) δ 22.3, 43.5, 64.9, 70.8, 75.1, 84.8, 91.2, 103.3, 144.6,157.4, 164.7, 166.5. ³¹ P NMR (CD₃ OD) δ 5.6. HRMS (FAB+) m/z 409.1119(Calc. 409.1124 for C₁₃ H₂₁ N₄ O₉ P+H⁺).

Compound 48. 5- N-(4-pyridylmethyl)carboxyamide!cytidine monophosphate.¹ H NMR (DMSO-d₆) δ 2.99 (q, J=7.3 Hz, 6H), 3.88 (m, 1H), 3.98 (m, 2H),4.03 (m, 1H), 4.13 (m, 1H), 4.32 (d, J=5.2 Hz, 2H), 5.92 (d, J=5.7 Hz,1H), 7.32 (d, J=5.4 Hz, 2H), 7.80 (s, 1H), 8.44 (d, J=4.8 Hz, 2H), 8.50(m, 1H), 8.79 (s, 1H), 10.38 (m, 1H). ¹³ C NMR (DMSO-d₆) δ 41.8, 63.9,70.4, 74.5, 83.7, 88.4, 98.6, 122.4, 144.0, 149.0, 149.2, 153.9, 162.3,163.7, 165.2. ³¹ P NMR (CD₃ OD) δ 6.5. HRMS (FAB+) m/z 458.1082 (Calc.458.1077 for C₁₆ H₂₀ N₅ O₉ P+H⁺).

Compound 49. 5- N-(2- 4-imidazole!ethyl)carboxyamide!cytidinemonophosphate. ¹ H NMR (1:1 D₂ O:CD₃ OD) δ 3.06 (m, 2H), 3.60 (m, 1H),3.68 (m, 1H), 4.12 (m, 2H), 4.25 (m, 1H), 4.29 (m, 2H), 5.95 (d, J=3.8Hz, 1H), 7.27 (s, 1H), 8.50 (s, 1H), 8.53 (s, 1H). ¹³ C NMR (1:1 D₂O:CD₃ OD) δ 27.9, 67.5, 73.8, 79.1, 88.0, 93.7, 104.7, 120.2, 135.9,136.8, 147.3, 159.3, 167.8, 170.1. ³¹ P (1:5 CD₃ OD:DMSO-d₆) δ 6.5. HRMS(FAB+) m/z 461.1186 (Calc. 461.1186 for C₁₅ H₂₁ N₆ O₉ P+H⁺).

Compound 50. 5- N-(2- 4-hydroxyphenyl!ethyl)carboxyamide!cytidinemonophosphate. ¹ H NMR (D₂ O) δ 2.82 (t, J=6.8 Hz, 2H), 3.52 (m, 2H),3.97 (m, 1H), 4.02 (m, 1H), 4.28 (m, 2H), 4.33 (t, J=4.6 Hz, 1H), 5.88(d, J=4.5 Hz, 1H), 6.83 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H), 8.24(s, 1H). ¹³ C NMR (D₂ O) δ 34.8, 42.0, 64.4, 70.9, 75.7, 85.0, 91.1,102.7, 116.2, 131.3, 132.3, 144.2, 155.1, 157.2, 164.7, 166.9. ³¹ P NMR(D₂ O) 7.8.

Compound 51. 5- N-(2-indolylethyl)carboxyamide!cytidine monophosphate. ¹H NMR (CD₃ OD) δ 2.94 (m, 2H), 3.55 (m, 2H), 4.04 (m, 1H), 4.11 (m, 2H),4.20 (m, 2H), 5.92 (d, J=4.2 Hz, 1H), 6.91 (m, 1H), 6.99 (m, 1H), 7.06(s, 1H), 7.25 (d, J=4.1 Hz, 1H), 7.61 (d, J=7.5 Hz, 1H), 8.56 (s, 1H).MS (FAB-) m/z (M-H)⁻ 507.7.

EXAMPLE 5 2'-Deoxycytidine Modifications with Amines

The following procedures were employed to produce the modified2'-deoxycytidines shown in Table IV. ##STR27##

                  TABLE IV                                                        ______________________________________                                        Summary of 2'-deoxycytidine carboxyamidation products.                                  Starting                Isolated Yield                              Entry     material                                                                              RNH.sub.2                                                                             Product ID                                                                            (%)                                         ______________________________________                                        1.sup.b   52      A       53      26                                          2.sup.a   52      B       54      20                                          ______________________________________                                         .sup.a Nucleotide (0.1 mmol), RNH.sub.2 (0.5 mmol), Pd.sub.2 (dba).sub.3      (2.5 μmol), P(pC.sub.6 H.sub.4 --SO.sub.3 Na).sub.3 (0.015 mmol), DABC     (0.7 mmol), DMSO (0.5 mL).                                                    .sup.b Nucleotide (0.1 mmol), RNH.sub.2 (0.5 mmol), Pd(PPh.sub.3).sub.4       (0.01 mmol), DBU or DABCO (0.5 mmol), DMSO:H.sub.2 O 93:7 (0.5 mL).           Compound 53. 5-(N-butylcarboxyamide)-2'-deoxycytidine monophosphate.     .sup.1 H NMR (D.sub.2 O) δ 0.90 (t, J=7.3 Hz, 3H), 1.35 (m, 2H),     1.58 (m, 2H), 2.30 (m, 1H), 2.52 (m, 1H), 3.31 (m, 2H), 4.11 (m, 2H), 4.26     (m, 1H), 4.54 (m, 1H), 6.22 (m, 1H), 8.41 (s, 1H).

Compound 54. 5- N-(2-4-hydroxyphenyl!ethyl)carboxyamide!-2'-deoxycytidine monophosphate. ¹ HNMR (D₂ O) δ 2.27 (m, 1H), 2.41 (m, 1H), 2.82 (m, 2H), 3.51 (m, 2H),3.90 (m, 2H), 4.19 (m, 1H), 4.50 (m, 1H), 6.16 (m, 1H), 6.83 (d, J=8.3Hz, 2H), 7.16 (d, J=8.3 Hz, 2H), 8.19 (s, 1H).

EXAMPLE 6 Determination of Antiviral Efficacy and Cellular Toxicity

This example demonstrates the ability of nucleotides of the invention toinhibit cytopathologic effects associated with human cytomagalovirus(CMV) infection. The experimental drugs of this example will be referredto as CT1146-26 and CT1146-28. CT1146-26 is Compound 47 and has thefollowing structure: ##STR28## CT1146-28 is Compound 45 and has thefollowing structure: ##STR29## Assay methodology and preliminary resultsare provided in the following sections.

Cell Isolation and Tissue Culture

Newborn human foreskins were obtained immediately post-circumcision andplaced in minimal essential medium (MEM) supplemented with vancomycin,fungizone, penicillin and gentamycin (all present at standardconcentrations) and maintained for four hours under tissue cultureconditions (37° C., 5% CO₂). Supplemented MEM was then removed and theforeskin material macerated. Tissue fragments were then washedexhaustively (using supplemented MEM) to remove residual erythrocytecontamination.

The erythrocyte-free cell fragments were then exposed to trypsinsolution (0.25%) for 15 min with continuous stirring. Tissue fragmentswere then allowed to settle out of suspension and the supernatantcollected and passed through sterile cheesecloth into a flask containingMEM and 10% fetal bovine serum (FBS). The cheesecloth was washed with anadditional volume of MEM containing 10% FBS (The flask containing MEMand 10% FBS was maintained on ice throughout the trypsinizationprocedure). The trypsinization procedure was repeated using freshtrypsin solution, until no additional cell removal from tissue fragmentswas observed. Cells removed by this process are predominantly humanforeskin fibroblasts (HFF).

Cells contained in MEM with 10% FBS were pelleted by centrifugation(≅1000 RPM, 4° C., 10 min), the supernatant discarded and the pelletedcells suspended in a minimal volume of MEM with 10% FBS. HFF isolateswere then plated into T-25 tissue culture flasks (the number of primaryculture flasks used was based on the volume of the recovered cellpellet). HFF isolates were then maintained under tissue cultureconditions (37° C., 5% CO₂) until confluent. Primary cultures of HFFwere sequentially expanded into larger format culture flasks usingstandard tissue culture procedures. HFF were maintained in the presenceof vancomycin and fungizone through passage four.

Cytotoxicity Assay: IC₅₀ Determination

HFF were seeded into 96-well tissue culture plates at a concentration of2.5×10⁴ cells/ml (100 μl MEM with 10% FBS was used as the culturemedium) and maintained under tissue culture conditions for 24 H prior toexperimentation. Medium from plates was then removed and 100 μl of MEMcontaining 2% FBS was added to all but the first row of cells in the96-well plate. To the first row of each 96-well plate, 125 μl of controlmedia or experimental drug CT1146-26 or CT1146-28 (initial concentrationdetermined by the overall concentration range desired) was added intriplicate wells. Medium alone was added to both cell and virus controlwells. The contents of the first row of wells were then serially diluted(1:5) across the remaining rows of the plate (25 μl volume transfer wellto well, with intermediate mixing, using a Cetus liquid handlingmachine). Following dilution, 100 μl of CMV (2500 PFU/well finalconcentration) in MEM with 2% FBS was added to each well of the 96-wellplate, except for wells containing cell controls. Cell control wellsreceived an additional 100 μl of MEM with 2% FBS. The 96-well plateswere then incubated under tissue culture conditions (14 day totalincubation period for CMV treated HFF in 2% FBS containing MEM; mediaaddition to cultures was made as appropriate).

Following the incubation period, medium was removed from all wells andthe cells stained with 0.1% crystal violet solution for 30 min followedby several wash cycles to remove residual stain. The crystal violetstained plates were then allowed to air dry for 24 H prior to readingwell absorbance values (620 nm) using a Skatron plate reader. Cellularviability and corresponding IC₅₀ values were determined based onabsorbance values for control and experimental drug treated cellsrelative to control cells which were not exposed to virus. IC₅₀ values(50% inhibitory concentration) for experimental drugs are determined asthe concentration of drug required to inhibit cellular proliferation by50%.

Plaque Reduction Assay Using Semi-Solid Overlay: EC₅₀ Determination

HFF are plated into 6-well tissue culture plates and maintained undertissue culture conditions for approximately two days prior to use. Onthe date of assay, experimental drug solutions are prepared as 2×concentrations in 2× MEM. Serial dilutions of experimental drug are thenperformed (1:5) using 2× MEM. The approximate concentration range forexperimental agents being 200 to 0.06 μg/ml. Each drug or controlsolution was then diluted 1:1 with 0.8% agarose solution. Followingdilution with agarose solution, the final experimental drugconcentration range was 100 to 0.03 μg/ml, with a final agarose overlayconcentration of 0.4%. Viral material (CMV) was diluted in MEM with 10%FBS to yield a concentration of virus producing 20-30 plaques per well.

Media was removed from HFF cultures and 200 μl of virus containing mediawas added to each well (200 μl of MEM was added to control wellscontaining cells not exposed to virus) of each 6-well plate. The assayplates were then incubated for 1 H with shaking every 15 min. Aliquots(2 ml) of agarose/experimental drug mixture were then applied induplicate to appropriate wells in 6-well culture plates. Control groupsreceived 2 ml aliquots of MEM/agarose in a 1:1 dilution. Platescontaining HFF and the various treatment groups were then incubatedunder tissue culture conditions for 14 days. On days 4 and 8, anadditional 1 ml of 1:1 2× MEM:agarose mixture was added to each well.

Following incubations, HFF were stained for 4-6 H with a 1.5% solutionof neutral red. The neutral red/agarose/MEM mixture is aspirated andviral plaques counted using a 10× steriomicroscope. EC₅₀ values (50%effective concentration) for each experimental drug are then determinedas the concentration of experimental drug required to inhibit viralcytopathogenicity by 50%.

The Selectivity Index (SI) for each drug treatment was also determined(SI=IC₅₀ /EC₅₀). Increasing efficacy of experimental drug in the absenceof equivalent cytotoxicity of the drug, will result in increasing SIratios (ie. A candidate compound with an IC₅₀ of 1 μg/ml and an EC₅₀ of0.01 μg/ml will have an SI=100; Conversely a candidate compound with anIC₅₀ of 0.01 μg/ml and an EC₅₀ of 1 μg/ml will have an SI=0.01).

Results

Initial studies using nucleosides CT1146-28 and CT1146-26 suggestsubstantive anti-viral activity with respect to CMV as shown in Table V:

                  TABLE V                                                         ______________________________________                                        Compound  EC.sub.50 (μg/ml)                                                                       IC.sub.50 (μg/ml)                                                                    SI                                           ______________________________________                                        CT1146-28 <0.03        >100      >3333                                        CT1146-26 <0.03        >100      >3333                                        ______________________________________                                         EC.sub.50 and IC.sub.50 values are against human cytomegalovirus (CMV)   

The novel nucleosides CT1146-28 and CT1146-26 exhibit significantanti-viral activity with respect to human CMV. Given the substantivedifferences between effective concentrations and cytotoxicity for theseagents, it is unlikely that observed results reflect de facto cellulartoxicity in response to treatment with these agents.

We claim:
 1. A method for the preparation of a modified nucleosidecomprising the steps of:reacting a cytosine containing a leaving groupattached to the 5- or 6-position of said cytosine with a nucleophile andcarbon monoxide in the presence of a palladium catalyst; and isolatingsaid modified nucleoside.
 2. The method of claim 1 wherein said cytosineis 5-halo-cytosine.
 3. The method of claim 2 wherein said5-halo-cytosine is selected from the group consisting of5-iodo-cytosine, 5-bromo-cytosine and 5-iodo-cytosine-5'-monophosphate.4. A method for the preparation of a modified nucleoside comprising thesteps of:reacting a uracil containing a leaving group attached to the 5-or 6-position of said uracil with a nucleophile and carbon monoxide inthe presence of a palladium catalyst; and isolating said modifiednucleoside.
 5. The method of claim 4 wherein said uracil is5-halo-uracil.
 6. The method of claim 5 wherein said 5-halo-uracil isselected from the group consisting of 5-iodo-uracil, 5-bromo-uracil and5-iodouracil-5'-monophosphate.
 7. The method of claim 6 wherein5-iodo-uracil is selected from the group consisting of5'-DMT-5-iodo-2',3'-O-isopropylideneuracil and5'-TBDMS-5-iodo-2',3'-O-isopropylideneuracil.
 8. A method for thepreparation of a modified nucleoside comprising the steps of:reacting anucleoside starting material containing a leaving group attached to the2-, 5-, 6- or 8-position of said nucleoside starting material with anucleophile and carbon monoxide in the presence of a palladium catalystwherein said nucleophile is selected from the group consisting of anamine, alcohol and thiol; and isolating said modified nucleoside.
 9. Themethod of claim 8 wherein said nucleophile has the formula RYH,wherein,Y is selected from the group consisting of O, S, NH and NR'; andR and R' are independently selected from the group consisting of aC1-C20 alkyl (straight chain or branched), C2-C20 alkenyl (straightchain or branched), aryl and an amino acid, wherein R and R' canoptionally be part of a cyclic structure which can be aromatic,aliphatic or heterocyclic.
 10. The method of claim 9 wherein R and R'are substituted with a functional group independently selected from thegroup consisting of an amide, ester, nitrile, nitro, urea halide,cyanate, alcohol, amine, ether, thiol and aryl.
 11. The method of claim9 wherein,Y is selected from the group consisting of O, S, and NH; and Ris (CH_(z))_(m) (CH₃)_(n), wherein z is 0, 1, or 2; m is 0-19; n is 0,1, 2, or 3; and wherein one or more of the H are optionally substitutedwith ═O, --OH, ═NH, NH₂, +NMe₃ Cl, ##STR30## or an amino acid.
 12. Themethod of claim 9 wherein said nucleophile is selected from the groupconsisting of: ##STR31##
 13. A method for the preparation of a modifiednucleoside comprising the steps of: reacting a nucleoside startingmaterial containing a leaving group attached to the 2-, 5-, 6- or8-position of said nucleoside starting material with a nucleophile andcarbon monoxide in the presence of a palladium catalyst of the formulaPdL₃ or PdL₄, wherein L is selected from the group consisting of P(C₆H₅)₃, P(p-C₆ H₄ SO₃ Na)₃, (o-tol)₃ P, CH₃ CN, DMSO,N,N-dimethylformamide (DMF), ##STR32## and isolating said modifiednucleoside.
 14. The method of claim 13 wherein said palladium catalystis Pd(P(C₆ H₅)₃)₄ or P(p-C₆ H₄ SO₃ Na)₃.
 15. A method for thepreparation of a modified nucleoside comprising the steps of:reacting anucleoside starting material containing a leaving group attached to the2-, 5-, 6- or 8-position of said nucleoside starting material with anucleophile and carbon monoxide in the presence of a palladium catalyst;wherein said leaving group is selected from the group consisting of ahalogen, acetate, trifluoroacetate, tosylate, methylsulfonate,trifluoromethyl sulfonate, boronic ester and boronic acid; and isolatingsaid modified nucleoside.
 16. The method of claim 15 wherein saidleaving group is a halogen.
 17. A method for the preparation of amodified nucleoside comprising the steps of:reacting a nucleosidestarting material containing a leaving group attached to the 2-, 5-, 6-or 8-position of said nucleoside starting material with a nucleophileand carbon monoxide in the presence of a palladium catalyst, in asolvent selected from the group consisting of THF, water, acetonitrile,dioxane, acetone, ethyl acetate, benzene, dimethyl formamide, dimethylacetamide, dimethyl sulfoxide, hexamethylphophoramide, andhexamethylphosphoroustriamide; and isolating said modified nucleoside.18. A compound selected from the group consisting of: ##STR33## wherein,Y is selected from the group consisting of O, S, NH and NR'; andR and R'are independently selected from the group consisting of C1-C20 alkyl(straight chain or branched), C2-C20 alkenyl (straight chain orbranched), aryl, heterocyclic, natural amino acids and unnatural aminoacids, wherein R and R' can optionally be part of a cyclic structurewhich can be aromatic, aliphatic, or heterocyclic; R" is selected fromthe group consisting of H and acyl; and Z is selected from the groupconsisting of a ribose, deoxyribose, dideoxyribose, and any combinationof 2', 3', and 5' modifications thereof.
 19. A compound of claim 18wherein,Y is selected from the group consisting of O, S, and NH; R is(CH_(z))_(m) (CH₃)_(n), wherein z is 0, 1, or 2; m is 0-19; n is 1, 2,or 3; and wherein one or more of the H are optionally substituted with═O, --OH, ═NH, NH₂, +NMe₃ Cl, ##STR34## or an amino acid.
 20. A compoundof claim 18 selected from the group consisting of ##STR35##
 21. Acompound of claim 18 selected from the group consisting of
 22. Thecompound of claim 18 incorporated as part of a oligonucleotide.
 23. Thecompound of claim 22 wherein said oligonucleotide is a ribonucleic acid.24. The compound of claim 22 wherein said oligonucleotide is adeoxyribonucleic acid.
 25. A method for inhibiting viral activitycomprising administering to a patient a therapeutically effective amountof a compound of claim
 18. 26. The method of claim 25 whereincytomegalovirus activity is inhibited.
 27. The method of claim 25wherein Y is NH and R is a C1-C20 alkyl.
 28. The method of claim 25wherein said compound is selected from the group consisting of